MODIFIED AMADORIASE AND METHOD FOR PRODUCING THE SAME, AGENT FOR IMPROVING SURFACTANT RESISTANCE OF AMADORIASE AND COMPOSITION FOR MEASURING HbA1c USING THE SAME

ABSTRACT

Provided is a composition by which glycated hemoglobin can be measured even in the presence of a stronger surfactant than a conventional case. Also provided is a buffer and/or stabilizer which maintains the residual activity of an amadoriase or lowers a reduction of residual activity. The present invention provides a composition for use in measuring glycated hemoglobin containing an amadoriase having substitution of one or more amino acid residues at a position(s) corresponding to an amino acid(s) selected from the group consisting of position 262, position 257, position 249, position 253, position 337, position 340, position 232, position 129, position 132, position 133, position 44, position 256, position 231 and position 81 of an amadoriase derived from the genus Coniochaeta and represented by SEQ ID No: 1 or 3, and having residual activity even in the presence of a surfactant. The present invention also provides a composition and kit for use in measuring glycated hemoglobin, comprising a specific stabilizer and/or a buffer. The present invention can provide an enzyme and a composition for use in measuring glycated hemoglobin, excellent in storage stability even if they are exposed to a surfactant.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.14/910,789, which is the U.S. National Stage of PCT/JP2014/071036, filedAug. 8, 2014, which claims priority to JP 2013-167005, filed Aug. 9,2013, and JP 2013-221515, filed Oct. 24, 2013.

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-WEB and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 24, 2020, isnamed sequence.txt and is 84,362 bytes.

TECHNICAL FIELD

The present invention relates to an amadoriase excellent in surfactantresistance, which can be advantageously used as a diagnostic enzyme fordiabetes and in a kit for measuring a diabetes marker, and relates to agene and recombinant DNA thereof and a method for producing anamadoriase excellent in surfactant resistance. The present inventionfurther relates to a stabilizer and/or a buffer for the amadoriase ofthe present invention and a composition containing the same.

Glycated proteins are generated by non-enzymatic covalent bondingbetween aldehyde groups in aldoses, such as glucose (monosaccharidespotentially containing aldehyde groups and derivatives thereof), andamino groups in proteins, followed by Amadori rearrangement. Examples ofamino groups in proteins include α-amino groups of the amino terminusand side chain ε-amino groups of the lysine residue in proteins.Examples of known glycated proteins generated in vivo include glycatedhemoglobin resulting from glycation of hemoglobin and glycated albuminresulting from glycation of albumin in the blood.

Among such glycated proteins generated in vivo, hemoglobin A1c (HbA1c)has drawn attention as a glycemic control marker significant fordiagnosis of diabetic patients and control of conditions in the field ofclinical diagnosis of diabetes mellitus. The blood HbA1c level reflectsthe average blood glucose level for a given period of time in the past,and the measured value thereof serves as a significant indicator fordiagnosis and control of diabetes conditions.

As a method for quickly and easily measuring HbA1c, an enzymatic methodusing an amadoriase is proposed, in which HbA1c is decomposed with e.g.,a protease, and α-fructosyl valyl histidine (hereinafter referred to as“αFVH”) or α-fructosyl valine (hereinafter referred to as “αFV”)released from a β chain amino terminus thereof is quantified (see, forexample, Patent Literatures 1 to 7). In reality, the method of cleavingαFV from HbA1c is associated with the problem in that accuratemeasurement values cannot be obtained since the effect of contaminantsand the like is significant. To obtain accurate measurement values, amethod of measuring αFVH is mainly employed in particular at present.

An amadoriase catalyzes a reaction of oxidizing iminodiacetic acid or aderivative thereof (also referred to as an “Amadori compound”) in thepresence of oxygen to produce glyoxylic acid or α-ketoaldehyde, an aminoacid or a peptide, and hydrogen peroxide.

Amadoriases have been found in bacteria, yeast, and fungi. Inparticular, amadoriases having enzyme activity to αFVH and/or αFV, whichare useful for measurement of HbA1c are for example, amadoriases derivedfrom the genera Coniochaeta, Eupenicillium, Pyrenochaeta, Arthrinium,Curvularia, Neocosmospora, Cryptococcus, Phaeosphaeria, Aspergillus,Emericella, Ulocladium, Penicillium, Fusarium, Achaetomiella,Achaetomium, Thielavia, Chaetomium, Gelasinospora, Microascus,Leptosphaeria, Ophiobolus, Pleospora, Coniochaetidium, Pichia,Debaryomyces, Corynebacterium, Agrobacterium, and Arthrobacter have beenreported (e.g., Patent Documents 1 and 6 to 15 and Non-Patent Documents1 to 11). In some of the aforementioned documents, amadoriase isoccasionally referred to as, for example, ketoamine oxidase, fructosylamino acid oxidase, fructosyl peptide oxidase, or fructosyl amineoxidase.

Regarding measurement of HbA1c, it is known that reagent compositionsfor measurement contain excessive amounts of amadoriase. For example,when measuring HbA1c at a final concentration of 0.36 μM, amadoriase isused at a concentration of 1.4 kU/L, which is a concentration at which1.4 mM of a substrate per minute can be reacted with the amadoriase (seePatent Literature 16). Measuring HbA1c using an amadoriase is currentlycarried out using an automated analyzer in the mainstream. Theamadoriase and substrate are often reacted for 5 minutes to 25 minutesand subjected to measurement therein. The reason for including excessiveamounts of amadoriase is to allow the amadoriase to react sufficientlywith the substrate during a short measurement time as mentioned above;and further, if a substance which has a negative effect on thereactivity and stability of the amadoriase is present in the compositionfor measurement, excessive amounts of amadoriase must be formulated as acountermeasure against the effect.

As a pretreatment for measuring HbA1c in whole blood or erythrocytes byusing an amadoriase, blood cells are lysed with a surfactant (see forexample, Patent Literatures 2, 16 to 18). When degrading HbA1c with aprotease, a surfactant is used in some methods as an accelerant (see,for example, Patent Literature 19). Therefore, surfactants areindispensable when measuring HbA1c with an amadoriase; however, thepossibility is extremely high for the surfactant to denature theamadoriase when an HbA1c solution, which is treated with a surfactantand a protease, is mixed with an amadoriase solution and then aquantitative reaction of HbA1c is started, as well as during storage ofa surfactant-amadoriase mixture. Presently used HbA1c measurement kitscontain excessive amounts of amadoriase than required, and further areformulated together with stabilizers and are able to achieve accuratemeasurement; however, the cost of the kit inevitably increases due touse of excessive reagents. Further, if it is possible to use a moreeffective surfactant than those presently used, the degradationefficiency of HbA1c with protease can be improved and it his highlypossible that the measurement sensitivity of HbA1c can be improved. Inaddition, surfactants have solubilizing effects on insoluble peptidefragments derived from hemoglobin and HbA1c. Because of the effect, thesurfactant can prevent turbidity, thereby contributing to improvement ofmeasurement accuracy. Therefore, regarding formulating an amadoriase asan enzyme for clinically diagnosing diabetes in a kit as a reagent, onedesirable property of the enzyme is to be stable in a liquid containinga surfactant.

Although individual measurement conditions vary; disclosure of thestability of various amadoriases in liquids can be found in literatureknown in the art: in a case where 5 mM ethylenediaminetetraacetic acidand 3% glycine are added in a solution containing an amadoriase derivedfrom Coniochaeta sp. NISL 9330 strain, it is reported that a residualactivity of 79% is maintained 7 days later at 30° C. (see, for example,Patent Literature 20). Further, in another case where 3% L-alanine, 3%glycine or 3% sarcosine is added in a solution containing a fructosylamino acid oxidase derived from Fusarium oxysporum IFO-9972 strain, itis reported that 100% residual activity is maintained 2 days later at37° C. (see, for example, Patent Literature 21).

However, no surfactants are added to the above solutions containingamadoriase protein and the literature is silent on reducing effects ofsurfactants. Furthermore, amadoriases having high surfactant resistancehave not been reported. Moreover, stabilizers and buffers maintainingthe residual activity of an amadoriase or lowering a reduction of theresidual activity in the presence of a surfactant have not beenreported.

PRIOR ART DOCUMENTS Patent Documents

-   Patent Document 1: WO 2004/104203-   Patent Document 2: WO 2005/49857-   Patent Document 3: JP 2001-95598 A-   Patent Document 4: JP H05-33997 B (1993)-   Patent Document 5: JP H11-127895 A (1999)-   Patent Document 6: WO 97/13872-   Patent Document 7: JP 2011-229526 A-   Patent Document 8: JP 2003-235585 A-   Patent Document 9: JP 2004-275013 A-   Patent Document 10: JP 2004-275063 A-   Patent Document 11: JP 2010-35469 A-   Patent Document 12: JP 2010-57474 A-   Patent Document 13: WO 2010/41715-   Patent Document 14: WO 2010/41419-   Patent Document 15: WO 2011/15325-   Patent Document 16: WO 2012/020744-   Patent Document 17: WO 2005/87946-   Patent Document 18: WO 2002/06519-   Patent Document 19: WO 2006/120976-   Patent Document 20: JP 2006-325547 A-   Patent Document 21: JP 2009-000128 A

Non-Patent Documents

-   Non-Patent Document 1: Biochem. Biophys. Res. Commun. 311, 104-11,    2003-   Non-Patent Document 2: Biotechnol. Bioeng. 106, 358-66, 2010-   Non-Patent Document 3: J. Biosci. Bioeng. 102, 241-3, 2006-   Non-Patent Document 4: Eur. J. Biochem. 242, 499-505, 1996-   Non-Patent Document 5: Arch. Microbiol. 178,344-50,2002-   Non-Patent Document 6: Mar. Biotechnol. 6,625-32, 2004-   Non-Patent Document 7: Biosci. Biotechnol. Biochem. 59, 487-91, 1995-   Non-Patent Document 8: Appl. Microbiol. Biotechnol. 74, 813-819,    2007-   Non-Patent Document 9: Biosci. Biotechnol. Biochem. 66, 1256-61,    2002-   Non-Patent Document 10: Biosci. Biotechnol. Biochem. 66, 2323-29,    2002-   Non-Patent Document 11: Biotechnol. Letters 27, 27-32, 2005

SUMMARY OF INVENTION Technical Problem

As described above, amadoriases have been used excessively in the art soas to sufficiently react with the substrate during measurement. Here,the present inventors newly found that a surfactant is a component whichsignificantly negatively effects the stability of amadoriases.Therefore, if an enzyme having more excellent surfactant resistance thanconventional amadoriases can be prepared, it is expected that such anenzyme will greatly contribute to attaining convenience in distribution(circulation) of the enzyme and kit, reduce amounts of the amadoriaseand stabilizers formulated in the kit, thereby lowering costs, andenable formulating strong surfactants, thereby improving measurementsensitivity of HbA1c. Therefore, an object of the present invention isto provide an amadoriase having excellent surfactant resistance comparedwith conventional amadoriases, as well as to provide a reagentcomposition by which HbA1c or a glycated peptide derived from HbA1c canbe quantified even in the presence of a surfactant.

Another object of the present invention is to provide a stabilizerand/or a buffer which maintains the residual activity of the amadoriaseor lowers reduction of the residual activity in the presence of asurfactant and a composition containing these.

Solution to Problem

Under the present situation where information regarding conferringsurfactant resistance to enzymes are barely disclosed, the presentinventors have conducted intensive studies. As a result, the presentinventors have found that the above objectives can be attained byintroducing a particular amino acid residue substitutions into anamadoriase derived from the genus Coniochaeta and further by formulatingto a reagent composition an amadoriase whose activity is retained evenin the presence of a surfactant. The present inventors further foundthat if a particular stabilizer and/or buffer is(are) used, the residualactivity of the amadoriase is maintained or reduction of residualactivity in the presence of a surfactant is significantly lowered. Basedon the findings, the present invention has been accomplished.

More specifically, the present invention is as follows.

1. An amadoriase having improved residual activity (%) 5 minutes after asurfactant is added compared with an amadoriase having an amino acidsequence as shown in SEQ ID No: 1, 3, or 37, and having

(i) an amino acid sequence having a deletion, insertion, addition,and/or substitution of one or several amino acids in the amino acidsequence as shown in SEQ ID No: 1, 3, or 37, and/or

(ii) an amino acid sequence having an identity of at least 70% with theamino acid sequence as shown in SEQ ID No: 1, 3, or 37.

2. The amadoriase according to [1], wherein the surfactant is an ionicsurfactant.3. The amadoriase according to [1] or [2], having substitution of one ormore amino acid residues at a position corresponding to an amino acidselected from the group consisting of the following (i) to (xiv):

(i) asparagine at position 262,

(ii) valine at position 257,

(iii) glutamic acid at position 249,

(iv) glutamic acid at position 253,

(v) glutamine at position 337,

(vi) glutamic acid at position 340,

(vii) aspartic acid at position 232,

(viii) aspartic acid at position 129

(ix) aspartic acid at position 132,

(x) glutamic acid at position 133,

(xi) glutamic acid at position 44

(xii) glycine at position 256,

(xiii) glutamic acid at position 231, and

(xiv) glutamic acid at position 81, in the amino acid sequence as shownin SEQ ID NO: 1 or 3.

4. The amadoriase according to any one of [1] to [3], wherein the aminoacids of the amino acid sequence as shown in SEQ ID NO: 1 or 3 have atleast one of the following substations (i) to (xiv):

(i) asparagine at position 262 is substituted with histidine;

(ii) valine at position 257 is substituted with cysteine, serine, orthreonine;

(iii) glutamic acid at position 249 is substituted with lysine, orarginine;

(iv) glutamic acid at position 253 is substituted with lysine, orarginine;

(v) glutamine at position 337 is substituted with lysine, or arginine;

(vi) glutamic acid at position 340 is substituted with proline;

(vii) aspartic acid at position 232 is substituted with lysine, orarginine;

(viii) aspartic acid at position 129 is substituted with lysine, orarginine;

(ix) aspartic acid at position 132 is substituted with lysine, orarginine;

(x) glutamic acid at position 133 is substituted with alanine,methionine, lysine, or arginine;

(xi) glutamic acid at position 44 is substituted with proline;

(xii) glycine at position 256 is substituted with lysine, or arginine;

(xiii) glutamic acid at position 231 is substituted with lysine, orarginine; and

(xiv) glutamic acid at position 81 is substituted with lysine, orarginine.

5. The amadoriase according to any one of [1] to [4], wherein the aminoacid sequence as shown in SEQ ID NO: 1 or 3 has substitution of aminoacid residues selected from the group consisting of the following (i) to(ix):

(i) substitution of an amino acid at the position corresponding toglutamic acid at position 44 with proline and substitution of an aminoacid at the position corresponding to glutamic acid at position 340 withproline;

(ii) substitution of an amino acid at the position corresponding toglutamic acid at position 44 with proline, substitution of an amino acidat the position corresponding to asparagine at position 262 withhistidine, and substitution of an amino acid at the positioncorresponding to glutamic acid at position 340 with proline;

(iii) substitution of an amino acid at the position corresponding toglutamic acid at position 44 with proline, substitution of an amino acidat the position corresponding to valine at position 257 with cysteine,substitution of an amino acid at the position corresponding toasparagine at position 262 with histidine, and substitution of an aminoacid at the position corresponding to glutamic acid at position 340 withproline;

(iv) substitution of an amino acid at the position corresponding toglutamic acid at position 44 with proline, substitution of an amino acidat the position corresponding to valine at position 257 with cysteine,substitution of an amino acid at the position corresponding toasparagine at position 262 with histidine, substitution of an amino acidat the position corresponding to glutamic acid at position 340 withproline, and substitution of an amino acid at the position correspondingto aspartic acid at position 232 with lysine;

(v) substitution of an amino acid at the position corresponding toglutamic acid at position 44 with proline, substitution of an amino-acidat the position corresponding to valine at position 257 with cysteine,substitution of an amino acid at the position corresponding toasparagine at position 262 with histidine, substitution of an amino acidat the position corresponding to glutamic acid at position 340 withproline, and substitution of an amino acid at the position correspondingto glutamic acid at position 249 with lysine;

(vi) substitution of an amino acid at the position corresponding toglutamic acid at position 44 with proline, substitution of an amino acidat the position corresponding to glutamic acid at position 253 withlysine, substitution of an amino acid at the position corresponding tovaline at position 257 with cysteine, substitution of an amino acid atthe position corresponding to asparagine at position 262 with histidine,and substitution of an amino acid at the position corresponding toglutamic acid at position 340 with proline;

(vii) substitution of an amino acid at the position corresponding toglutamic acid at position 44 with proline, substitution of an amino acidat the position corresponding to glutamic acid at position 253 withlysine, substitution of an amino acid at the position corresponding tovaline at position 257 with cysteine, substitution of an amino acid atthe position corresponding to asparagine at position 262 with histidine,substitution of an amino acid at the position corresponding to glutamicacid at position 340 with proline, and substitution of an amino acid atthe position corresponding to aspartic acid at position 129 with lysine.

(viii) substitution of an amino acid at the position corresponding toglutamic acid at position 44 with proline, substitution of an amino acidat the position corresponding to glutamic acid at position 133 withalanine, substitution of an amino acid at the position corresponding toglutamic acid at position 253 with lysine, substitution of an amino acidat the position corresponding to valine at position 257 with cysteine,substitution of an amino acid at the position corresponding toasparagine at position 262 with histidine, and substitution of an aminoacid at the position corresponding to glutamic acid at position 340 withproline; and

(ix) substitution of an amino acid at the position corresponding toglutamic acid at position 44 with proline, substitution of an amino acidat the position corresponding to glutamic acid at position 133 withalanine, substitution of an amino acid at the position corresponding toglutamic acid at position 253 with lysine, substitution of an amino acidat the position corresponding to valine at position 257 with cysteine,substitution of an amino acid at the position corresponding toasparagine at position 262 with histidine, substitution of an amino acidat the position corresponding to glutamine at position 337 with lysine,and substitution of an amino acid at the position corresponding toglutamic acid at position 340 with proline.

6. The amadoriase according to any one of [1] to [3], wherein aminoacids of the amino acid sequence as shown in SEQ ID NO: 37 have at leastone of the substitutions of the following (i) to (ix)

(i) glutamic acid at position 247 is substituted with lysine, orarginine;

(ii) glutamic acid at position 251 is substituted with lysine, orarginine;

(iii) threonine at position 335 is substituted with lysine, or arginine;

(iv) aspartic acid at position 230 is substituted with lysine, orarginine;

(v) aspartic acid at position 129 is substituted with lysine, orarginine;

(vi) aspartic acid at position 132 is substituted with lysine, orarginine;

(vii) glutamic acid at position 133 is substituted with alanine,methionine, lysine, or arginine;

(viii) asparagine at position 254 is substituted with lysine, orarginine; and

(ix) glutamic acid at position 229 is substituted with lysine, orarginine.

7. The amadoriase according to any one of [1] to [3] and [6], whereinthe amino acid sequence as shown in SEQ ID NO: 37 has substitution ofamino acid residues selected from the group consisting of the following(i) to (iv):

(i) substitution of an amino acid at the position corresponding toglutamic acid at position 251 with lysine and substitution of an aminoacid at the position corresponding to threonine at position 335 withlysine;

(ii) substitution of an amino acid at the position corresponding toaspartic acid at position 132 with lysine and substitution of an aminoacid at the position corresponding to threonine at position 335 withlysine;

(iii) substitution of an amino acid at the position corresponding toglutamic acid at position 133 with alanine and substitution of an aminoacid at the position corresponding to threonine at position 335 withlysine; and

(iv) substitution of an amino acid at the position corresponding toglutamic acid at position 229 with lysine and substitution of an aminoacid at the position corresponding to threonine at position 335 withlysine.

8. An amadoriase gene encoding the amino acid sequence according to anyone of [1] to [7].9. A recombinant vector comprising the amadoriase gene according to [8].10. A host cell comprising the recombinant vector according to [9].11. A method for producing an amadoriase comprising the following steps:

(i) culturing the host cell according to [10];

(ii) expressing an amadoriase gene contained in the host cell; and

(iii) isolating the amadoriase from a culture product.

12. A composition comprising the amadoriase according to any one of [1]to [7], for use in measuring glycated hemoglobin.13. A composition comprising one or more surfactants and an amadoriasefor measuring glycated hemoglobin.14. The composition according to [13], wherein the amadoriase

(i) has a residual activity (%) of 15% or higher 5 minutes after asurfactant is added compared with a case where no surfactant is added,and/or

(ii) exhibits a difference of 0.006 or higher between absorbance at 751nm after a colorimetric substrate sodiumN-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylamine(DA-64) is added and reacted for 5 minutes, and absorbance at 751 nm 5minutes after a control solution containing ion-exchanged water in placeof a glycated amino acid solution or a glycated peptide solution isadded, in the presence of a 0.04% final concentration of surfactant.

15. The composition according to [13] or [14], wherein the amadoriasehas an amino acid sequence having an identity of at least 70% with theamino acid sequence as shown in SEQ ID No: 1, 3, 37 or 40.16. The composition according to any one of [13] to [15], wherein thesurfactant has a critical micelle concentration of 70 mM or lower.17. The composition according to any one of [13] to [16], wherein thesurfactant is one or more ionic surfactants selected from the groupconsisting of a quaternary ammonium salt represented by the followinggeneral formula (I):

[Formula 1]

[wherein, R¹ to R⁴, which may be the same or different, each represent asubstituted or unsubstituted C₁ to C₂₀ alkyl, alkenyl, aryl or benzyl;and Z⁻ represents a monovalent anion], a pyridinium salt represented bythe following general formula (II):

[wherein, R⁵ represents a substituted or unsubstituted C₁ to C₂₀ alkyl,a plurality of R^(a), which may be the same or different, each representa hydrogen atom or a substituted or unsubstituted C₁ to C₂₀ alkyl,alkenyl, aryl or benzyl; n represents an integer of 1 to 5; and Z⁻represents a monovalent anion],a phosphonium salt represented by the general formula (III),

[wherein, R⁶ to R⁹, which may be the same or different, each represent asubstituted or unsubstituted C₁ to C₂₀ alkyl, alkenyl, aryl or benzyl;and Z⁻ represents a monovalent anion]. and sodium dodecyl sulfate.18. The composition according to [17], wherein the surfactant is one ormore ionic surfactants selected from the group consisting of

octyltrimethylammonium chloride, octyltrimethylammonium bromide,dioctyldimethylammonium chloride, dioctyldimethylammonium bromide,decyltrimethylammonium chloride, decyltrimethylammonium bromide,dodecyltrimethylammonium chloride, dodecyltrimethylammonium bromide,tetradecyltrimethylammonium chloride, tetradecyltrimethylammoniumbromide, hexadecyltrimethylammonium chloride, hexadecyltrimethylammoniumbromide, octadecyltrimethylammonium chloride, octadecyltrimethylammoniumbromide, eicosyltrimethylammonium chloride and eicosyltrimethylammoniumbromide, benzyldodecyldimethylammonium chloride,benzyldodecyldimethylammonium bromide, benzyltetradecyldimethylammoniumchloride, benzyltetradecyldimethylammonium bromide,benzylcetyldimethylammonium chloride, and benzylcetyldimethylammoniumbromide,

1-decylpyridinium chloride, 1-decylpyridinium bromide,1-dodecylpyridinium chloride, 1-dodecylpyridinium bromide,1-tetradecylpyridinium chloride, 1-tetradecylpyridinium bromide,1-hexadecylpyridinium chloride, 1-hexadecylpyridinium bromide,N-cetyl-2-methylpyridinium chloride, N-cetyl-2-methylpyridinium bromide,N-cetyl-3-methylpyridinium chloride, N-cetyl-3-methylpyridinium bromide,N-cetyl-4-methylpyridinium chloride, N-cetyl-4-methylpyridinium bromide,1-octadecylpyridinium chloride, 1-octadecylpyridinium bromide,1-eicosylpyridinium chloride and 1-eicosylpyridinium bromide,

tetraethylphosphonium chloride, tetraethylphosphonium bromide,tributylmethylphosphonium chloride, tributylmethylphosphonium bromide,tributylmethylphosphonium iodide, tetrabutylphosphonium chloride,tetrabutylphosphonium bromide, tetra-n-octylphosphonium chloride,tetra-n-octylphosphonium bromide, tributyldodecylphosphonium chloride,tributyldodecylphosphonium bromide, tributylhexadecylphosphoniumchloride, tributylhexadecylphosphonium bromide,methyltriphenylphosphonium chloride, methyltriphenylphosphonium bromide,methyltriphenylphosphonium iodide, tetraphenylphosphonium chloride andtetraphenylphosphonium bromide.

19. The composition according to any one of [13] to [18], wherein thesurfactant contained has a final concentration of 0.01% (w/v) or higherat the time of measurement.20. The composition for use in measuring glycated hemoglobin accordingto [13], further comprising one or more buffers selected from the groupconsisting of

a borate buffer, a Tris-HCl buffer, a phosphate buffer, a citratebuffer, a fumarate buffer, a glutarate buffer, a citraconate buffer, amesaconate buffer, a malonate buffer, a tartrate buffer, a succinatebuffer, an adipate buffer, ACES (N-(2-acetamido)-2-aminoethanesulfonicacid) buffer, BES (N,N-bis(2-hydroxyethyl)-2-amino-ethanesulfonic acid)buffer, Bicin (N,N-bis(2-hydroxyethyl)glycine) buffer, Bis-Tris(bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane) buffer, EPPS(4-(2-hydroxyethyl)-1-piperazine-propanesulfonic acid) buffer, HEPPSO(N-(hydroxyethyl)piperazine-N′-2-hydroxypropanesulfonic acid) buffer,MES (2-(n-morpholino)ethanesulfonic acid) buffer, MOPS(3-(N-morpholino)propanesulfonic acid) buffer, MOPSO(2-hydroxy-3-morpholino-propanesulfonate) buffer, PIPES(piperazine-N,N′-bis(2-ethanesulfonic acid)) buffer, POPSO(piperazine-1,4-bis(2-hydroxypropanesulfonic acid)) buffer, TAPS(N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid) buffer, TAPSO(3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid)buffer, TES (N-tris(hydroxymethyl)methyl-2-amino-ethanesulfonic acid)buffer, Tricine (N-Tris(hydroxymethyl)methylglycine) buffer and acombination thereof.

21. The composition according to [20], comprising one or more buffersselected from the group consisting of a phosphate buffer having a finalconcentration in the measurement solution of 100 mM or higher, a citratebuffer having a final concentration in the measurement solution of 10 mMor higher, MES (2-(n-morpholino)ethanesulfonic acid) buffer having afinal concentration in the measurement solution of 150 mM or higher,MOPS (3-(n-morpholino)propanesulfonic acid) buffer having a finalconcentration in the measurement solution of 100 mM or higher MOPSO(2-hydroxy-3-morpholino-propanesulfonic acid) buffer having a finalconcentration in the measurement solution of 100 mM or higher, and ACES(N-(2-acetamido)-2-aminoethanesulfonic acid) buffer having a finalconcentration in the measurement solution of 200 mM or higher.22. The composition for use in measuring glycated hemoglobin accordingto [13], further comprising one or more stabilizers selected from thegroup consisting of phosphoric acid, a tricarboxylic acid, adicarboxylic acid, a monocarboxylic acid, a compound represented by theformula (IV)

[wherein, n may be 0, 1, 2 or 3; and R¹⁰ each independently representsH, OH, —CH₂OH or —COOH],ammonium sulfate and a combination thereof.23. The composition according to [22], wherein the tricarboxylic acid iscitric acid, or the dicarboxylic acid is selected from the groupconsisting of fumaric acid, glutaric acid, citraconic acid, mesaconicacid, malonic acid, tartaric acid, succinic acid, adipic acid, maleicacid, malic acid and combination of these; the mono-carboxylic acid isacetic acid; or the compound represented by the formula (IV) is selectedfrom the group consisting of MES, MOPS, MOPSO and a combination thereof.24. The composition according to [22] or [23], wherein

the stabilizer is one or more stabilizers selected from the groupconsisting of phosphoric acid having a final concentration in themeasurement solution of 2 mM or higher, citric acid having a finalconcentration in the measurement solution of 0.2 mM or higher, malicacid having a final concentration in the measurement solution of 2 mM orhigher, maleic acid having a final concentration in the measurementsolution of 2 mM or higher, citraconic acid having a final concentrationin the measurement solution of 2 mM or higher, malonic acid having afinal concentration in the measurement solution of 2 mM or higher,glutaric acid having a final concentration in the measurement solutionof 2 mM or higher, tartaric acid having a final concentration in themeasurement solution of 2 mM or higher, acetic acid having a finalconcentration in the measurement solution of 10 mM or higher, MES(2-(n-morpholino)ethanesulfonic acid) having a final concentration inthe measurement solution of 10 mM or higher, MOPS(3-(n-morpholino)propanesulfonic acid) having a final concentration inthe measurement solution of 10 mM or higher, MOPSO(2-hydroxy-3-morpholinopropanesulfonic acid) having a finalconcentration in the measurement solution of 10 mM or higher, ammoniumsulfate having a final concentration in the measurement solution of 2 mMor higher and a combination thereof.

25. A composition for use in measuring glycated hemoglobin, comprisingthe buffer according to [20] or [21] and the stabilizer according to[22], [23] or [24].26. The composition according to any one of [20] to [25], wherein theamadoriase is an amadoriase having an amino acid sequence represented bySEQ ID No: 1, SEQ ID No: 37 or SEQ ID No: 40 or the amadoriase accordingto any one of [1] to [7].

The specification incorporates the contents described in thespecifications and/or drawings described in JP Patent Application Nos.2013-167005 and 2013-221515, based on which the priority of thisapplication is claimed.

Advantageous Effects of Invention

According to the present invention, it is possible to provide anamadoriase excellent in surfactant resistance which can advantageouslybe used as a diagnostic enzyme for diabetes in a kit for measuring adiabetes marker, as well as a gene encoding the amadoriase and the like.Use of the amadoriase enables measurement of glycated hemoglobin even inthe presence of high concentrations of surfactants. Further, use of thestabilizer and/or buffer of the present invention enables retaining theresidual activity of an amadoriase or lowering reduction of the residualactivity in the presence of a surfactant, as well as measuring glycatedhemoglobin even in the presence of a surfactant at high concentrations.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A shows alignment of amino acid sequences of various amadoriasesknown in the art.

FIG. 1B shows alignment of amino acid sequences of various amadoriasesknown in the art.

FIG. 1C shows alignment of amino acid sequences of various amadoriasesknown in the art.

FIG. 2 shows the measurement results of αFVH by using CFP-T7 in thepresence of 0.01% CTAC in a mixture.

FIG. 3 shows the measurement results of αFVH by using CFP-T7 in thepresence of 0.02% CTAC in a mixture.

FIG. 4 shows the measurement results of αFVH by using CFP-D7 in thepresence of 0.02% CTAC in a mixture.

FIG. 5 shows the measurement results of αFVH by using CFP-D7 in thepresence of 0.2% CTAC in a mixture.

DESCRIPTION OF EMBODIMENTS

The present invention is described below in detail.

(Amadoriase)

Amadoriase, which is referred to also as e.g., ketoamine oxidase,fructosyl amino acid oxidase, fructosyl peptide oxidase or fructosylamine oxidase, refers to an enzyme which catalyzes a reaction ofoxidizing iminodiacetic acid or a derivative thereof (Amadori compound)in the presence of oxygen to generate glyoxylic acid or α-ketoaldehyde,an amino acid or a peptide and hydrogen peroxide. Amadoriases are widelydistributed in nature and can be obtained by searching microorganism-,animal- or plant-derived enzymes. Regarding microorganisms, anamadoriase can be obtained from, for example, filamentous fungi, yeastor bacteria.

An aspect of the amadoriase of the present invention is directed to anamadoriase variant having improved surfactant resistance, which isproduced based on an amadoriase derived from Coniochaeta having theamino acid sequenceas shown in SEQ ID NO: 1 or an amadoriase derivedfrom Curvularia clavata having the amino acid sequence as shown in SEQID NO: 37. Examples of such variant include an amadoriase having anamino acid sequence having a high sequence identity (for example, 70% orhigher, preferably 75% or higher, preferably 80% or higher, morepreferably 85% or higher, further preferably 90% or higher, furtherpreferably 95% or higher, further preferably 97% or higher, mostpreferably 99% or higher) with the amino acid sequence as shown in SEQID NO: 1 or SEQ ID No: 37; as well as an amadoriase having an amino acidsequence as shown in SEQ ID NO: 1 or SEQ ID No: 37, in which a single orseveral amino acids is(are) modified or mutated, in other words,deleted, substituted, added and/or inserted.

The amadoriase of the present invention may be prepared based on anamadoriase derived from any one of organism species such as the generaEupenicillium, Pyrenochaeta, Arthrinium, Curvularia, Neocosmospora,Cryptococcus, Phaeosphaeria, Aspergillus, Emericella, Ulocladium,Penicillium, Fusarium, Achaetomiella, Achaetomium, Thielavia,Chaetomium, Gelasinospora, Microascus, Leptosphaeria, Ophiobolus,Pleospora, Coniochaetidium, Pichia, Corynebacterium, Agrobacterium andArthrobacter. Among these, an amadoriase having surfactant resistanceand/or having an amino acid sequence which has high sequence identitywith that as shown in SEQ ID NO: 1 or SEQ ID No: 37, is preferable.

An amadoriase variant (modified amadoriase) having a modified surfactantresistance can be obtained by substituting, adding or deleting at leastone amino acid residue of the amino acid sequence of an amadoriase.

As amino acid substitutions which provide improvement of surfactantresistance, substitutions of amino acids at the positions correspondingto the amino acids at the following positions in the amino acid sequenceas shown in SEQ ID NO: 1 or 3, are mentioned.

(1) substitution of asparagine at position 262 with, e.g., histidine.

(2) substitution of valine at position 257 with, e.g., cysteine, serine,threonine.

(3) substitution of glutamic acid at position 249 with, e.g., lysine,arginine.

(4) substitution of glutamic acid at position 253 with, e.g., lysine,arginine.

(5) substitution of glutamine at position 337 with, e.g., lysine,arginine.

(6) substitution of glutamic acid at position 340 with, e.g., proline.

(7) substitution of aspartic acid at position 232 with, e.g., lysine,arginine.

(8) substitution of aspartic acid at position 129 with, e.g., lysine,arginine.

(9) substitution of aspartic acid at position 132 with, e.g., lysine,arginine.

(10) substitution of glutamic acid at position 133 with, e.g., alanine,methionine, lysine, arginine.

(11) substitution of glutamic acid at position 44 with, e.g., proline.

(12) substitution of glycine at position 256 with, e.g., lysine,arginine.

(13) substitution of glutamic acid at position 231 with, e.g., lysine,arginine.

(14) substitution of glutamic acid at position 81 with, e.g., lysine,arginine.

The amadoriase variant with improved surfactant resistance may have atleast one of the above mentioned amino-acid substitutions and may have aplurality of amino-acid substitutions. The amadoriase variant has, forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 of the aboveamino-acid substitutions.

Among such variants, those having amino-acid substitutions correspondingto the following amino acid positions are preferable.

(11)-(6) a variant having substitution of glutamic acid at position 44and substitution of glutamic acid at position 340, for example,substitution of an amino acid at the position corresponding to glutamicacid at position 44 with proline, and substitution of an amino acid atthe position corresponding to glutamic acid at position 340 withproline.

(11)-(1)-(6) a variant having substitution of glutamic acid at position44, substitution of asparagine at position 262 and substitution ofglutamic acid at position 340, for example, substitution of an aminoacid at the position corresponding to glutamic acid at position 44 withproline, substitution of an amino acid at the position corresponding toasparagine at position 262 with histidine, and substitution of an aminoacid at the position corresponding to glutamic acid at position 340 withproline.

(11)-(2)-(1)-(6) a variant having substitution of glutamic acid atposition 44, substitution of valine at position 257, substitution ofasparagine at position 262 and substitution of glutamic acid at position340, for example, substitution of an amino acid at the positioncorresponding to glutamic acid at position 44 with proline, substitutionof an amino acid at the position corresponding to valine at position 257with cysteine, substitution of an amino acid at the positioncorresponding to asparagine at position 262 with histidine, andsubstitution of an amino acid at the position corresponding to glutamicacid at position 340 with proline.

(11)-(7)-(2)-(1)-(6) a variant having substitution of glutamic acid atposition 44, substitution of valine at position 257, substitution ofasparagine at position 262, substitution of glutamic acid at position340 and substitution of aspartic acid at position 232, for example,substitution of an amino acid at the position corresponding to glutamicacid at position 44 with proline, substitution of an amino acid at theposition corresponding to valine at position 257 with cysteine,substitution of an amino acid at the position corresponding toasparagine at position 262 with histidine, substitution of an amino acidat the position corresponding to glutamic acid at position 340 withproline, and substitution of an amino acid at the position correspondingto aspartic acid at position 232 with lysine or arginine.

(11)-(3)-(2)-(1)-(6) a variant having substitution of glutamic acid atposition 44, substitution of valine at position 257, substitution ofasparagine at position 262, substitution of glutamic acid at position340 and substitution of glutamic acid at position 249, for example,substitution of an amino acid at the position corresponding to glutamicacid at position 44 with proline, substitution of an amino acid at theposition corresponding to valine at position 257 with cysteine,substitution of an amino acid at the position corresponding toasparagine at position 262 with histidine, substitution of an amino acidat the position corresponding to glutamic acid at position 340 withproline, and substitution of an amino acid at the position correspondingto glutamic acid at position 249 with lysine or arginine.

(11)-(4)-(2)-(1)-(6) a variant having substitution of glutamic acid atposition 44, substitution of glutamic acid at position 253, substitutionof valine at position 257, substitution of asparagine at position 262and substitution of glutamic acid at position 340, for example,substitution of an amino acid at the position corresponding to glutamicacid at position 44 with proline, substitution of an amino acid at theposition corresponding to glutamic acid at position 253 with lysine orarginine, substitution of an amino acid at the position corresponding tovaline at position 257 with cysteine, substitution of an amino acid atthe position corresponding to asparagine at position 262 with histidineand substitution of an amino acid at the position corresponding toglutamic acid at position 340 with proline.

(11)-(8)-(4)-(2)-(1)-(6) a variant having substitution of glutamic acidat position 44, substitution of glutamic acid at position 253substitution of valine at position 257, substitution of asparagine atposition 262, substitution of glutamic acid at position 340 andsubstitution of aspartic acid at position 129, for example, substitutionof an amino acid at the position corresponding to glutamic acid atposition 44 with proline, substitution of an amino acid at the positioncorresponding to glutamic acid at position 253 with lysine or arginine,substitution of an amino acid at the position corresponding to valine atposition 257 with cysteine, substitution of an amino acid at theposition corresponding to asparagine at position 262 with histidine,substitution of an amino acid at the position corresponding to glutamicacid at position 340 with proline, and substitution of an amino acid atthe position corresponding to aspartic acid at position 129 with lysineor arginine.

(11)-(10)-(4)-(2)-(1)-(6) a variant having substitution of glutamic acidat position 44, substitution of glutamic acid at position 133,substitution of glutamic acid at position 253, substitution of valine atposition 257, substitution of asparagine at position 262 andsubstitution of glutamic acid at position 340, for example, substitutionof an amino acid at the position corresponding to glutamic acid atposition 44 with proline, substitution of an amino acid at the positioncorresponding to glutamic acid at position 133 with alanine,substitution of an amino acid at the position corresponding to glutamicacid at position 253 with lysine or arginine, substitution of an aminoacid at the position corresponding to valine at position 257 withcysteine, substitution of an amino acid at the position corresponding toasparagine at position 262 with histidine, and substitution of an aminoacid at the position corresponding to glutamic acid at position 340 withproline.

(11)-(10)-(4)-(2)-(1)-(5)-(6) a variant having substitution of glutamicacid at position 44, substitution of glutamic acid at position 133,substitution of glutamic acid at position 253, substitution of valine atposition 257, substitution of asparagine at position 262, substitutionof glutamine at position 337 with lysine, and substitution of glutamicacid at position 340, for example, substitution of an amino acid at theposition corresponding to glutamic acid at position 44 with proline,substitution of an amino acid at the position corresponding to glutamicacid at position 133 with alanine, substitution of an amino acid at theposition corresponding to glutamic acid at position 253 with lysine orarginine, substitution of an amino acid at the position corresponding tovaline at position 257 with cysteine, substitution of an amino acid atthe position corresponding to asparagine at position 262 with histidine,substitution of an amino acid at the position corresponding to glutamineat position 337 with lysine or arginine, and substitution of an aminoacid at the position corresponding to glutamic acid at position 340 withproline.

The amadoriase variant excellent in surfactant resistance according tothe present invention may have an amino-acid substitution(s) asmentioned above, which provides improved surfactant resistance to theamadoriase, regarding the amino acid sequence as shown in SEQ ID No: 1.The surfactant-resistant amadoriase variant of the present invention mayfurther have a deletion, insertion, addition and/or substitution of asingle or several amino acids (for example, 1 to 15 amino acids, 1 to 10amino acids, preferably 1 to 5 amino acids, further preferably 1 to 3amino acids, particularly preferably a single amino acid) at thepositions excluding the positions of substituted amino acids. Further,the present invention encompasses an amadoriase variant with modifiedsurfactant resistance, comprising an amino-acid substitution mutationproviding improved surfactant resistance as mentioned above and anamino-acid substitution mutation providing improved properties otherthan surfactant resistance, such as substrate specificity and the like;wherein said variant has an amino acid sequence identity of 70% orhigher, 75% or higher, 80% or higher, 90% or higher, further preferably95% or higher, further preferably 97% or higher and particularlypreferably 99% or higher, with the amino acid sequences as shown in SEQID NO: 1 or 3 albeit excluding those amino acids at the aforementionedamino-acid substitutions, and having an amadoriase activity.

The amadoriase having the amino acid sequence as shown in SEQ ID NO: 1is an amadoriase (CFP-T7) derived from the genus Coniochaeta produced byEscherichia coli (deposition number: FERM BP-10593) having a recombinantplasmid designated as pKK223-3-CFP-T7 in WO2007/125779, and this is amodified amadoriase having excellent thermal stability previously foundby the applicant. CFP-T7 is a triple variant obtained by sequentiallyintroducing artificial mutations into a native amadoriase derived fromthe genus Coniochaeta, at positions 272, 302 and 388.

SEQ ID No: 3 represents the amino acid sequence of an amadoriase derivedfrom the genus Coniochaeta obtained by introducing a mutation forimproving substrate specificity (E98A) disclosed in WO2012/18094 andmutations for enhancing heat stability (F43Y, G184D, deletion of threeamino acid residues in the carboxyl terminus) disclosed in WO2007/125779 and WO2013/100006.

In the above amino-acid substitutions, the positions of amino acidsrepresent the positions in the amino acid sequence of the amadoriasederived from the genus Coniochaeta and shown in SEQ ID No: 1. In theamino acid sequences of amadoriases derived from other species, theamino acids of the corresponding positions to the positions in the aminoacid sequence as shown in SEQ ID NO: 1 are substituted. The meaning ofthe phrase “corresponding position(s)” will be described later.

As the amino-acid substitution providing improvement of surfactantresistance, amino-acid substitutions at the positions corresponding toamino acids at the following positions in the amino acid sequence asshown in SEQ ID No: 37 are mentioned.

(i) substitution of glutamic acid at position 247, e.g., substitutionwith lysine, arginine;

(ii) substitution of glutamic acid at position 251, e.g., substitutionwith lysine, arginine;

(iii) substitution of threonine at position 335, e.g., substitution withlysine, arginine;

(iv) substitution of aspartic acid at position 230, e.g., substitutionwith lysine, arginine;

(v) substitution of aspartic acid at position 129, e.g., substitutionwith lysine, arginine;

(vi) substitution of aspartic acid at position 132, e.g., substitutionwith lysine, arginine;

(vii) substitution of glutamic acid at position 133, e.g., alanine,methionine, lysine, arginine;

(viii) substitution of asparagine at position 254, e.g., substitutionwith lysine, arginine; and

(ix) substitution of glutamic acid at position 229, e.g., substitutionwith lysine, arginine.

An amadoriase variant with improved surfactant resistance may have atleast one of the above amino-acid substitutions or may have a pluralityof amino-acid substitutions. For example, the amadoriase variant has 1,2, 3, 4, 5, 6, 7, 8, or 9 of the above amino-acid substitutions.

Among them, variants having amino acid substitutions at the positionscorresponding to the following amino acid positions are preferable; morespecifically, the following (i) to (iv) in the amino acid sequence asshown in SEQ ID No: 37:

(i) a variant having substitution of an amino acid at the positioncorresponding to glutamic acid at position 251 with lysine andsubstitution of an amino acid at the position corresponding to threonineat position 335 with lysine;

(ii) a variant having substitution of an amino acid at the positioncorresponding to aspartic acid at position 132 with lysine andsubstitution of an amino acid at the position corresponding to threonineat position 335 with lysine;

(iii) a variant having substitution of an amino acid at the positioncorresponding to glutamic acid at position 133 with alanine andsubstitution of an amino acid at the position corresponding to threonineat position 335 with lysine; and

(iv) a variant having substitution of an amino acid at the positioncorresponding to glutamic acid at position 229 with lysine andsubstitution of an amino acid at the position corresponding to threonineat position 335 with lysine.

(Obtaining a Gene Encoding an Amadoriase)

In order to obtain a gene in accordance with the present inventionencoding these amadoriases (hereinafter, also referred to as merely“amadoriase gene”), gene cloning methods used in general can be carriedout. For example, chromosomal DNA or mRNA can be extracted from amicroorganism fungus body or various cells having an ability to producean amadoriase by a conventional technique, such as a method described in“Current Protocols in Molecular Biology” (WILEY Interscience, 1989). Inaddition, cDNA can be synthesized using mRNA as a template. Achromosomal DNA or cDNA library can be made using the chromosomal DNA orcDNA obtained in such a manner.

Subsequently, DNA including the entire sequence of a target amadoriasegene can be obtained by a method of synthesizing an appropriate probeDNA based on the amino acid sequence of the aforementioned amadoriaseand selecting an amadoriase gene from a chromosomal DNA or cDNA libraryusing the probe DNA. Alternatively, an appropriate primer DNA may beproduced based on the aforementioned amino acid sequence, a DNAincluding the target gene fragment encoding the amadoriase gene may beamplified by using an appropriate PCR technique, such as the 5′ RACE or3′ RACE method, and the resulting DNA fragments may then be linked.

A preferable example of a gene encoding an amadoriase thus obtained isan example of an amadoriase gene derived from the genus Coniochaeta (JP2003-235585 A) among others.

Such amadoriase genes are preferably linked to various vectors accordingto a conventional technique from the viewpoint of handleability. Oneexample may be the recombinant plasmid pKK223-3-CFP (JP 2003-235585 A)in which the DNA encoding an amadoriase gene derived from theConiochaeta sp. NISL9330 strain has been inserted into the pKK223-3vector (GE Healthcare).

(Vector)

Vectors that can be used in the present invention are not limited to theaforementioned plasmid vectors and include, for example, any othervectors known in the art, such as bacteriophage or cosmid vectors. Morespecifically, for example, pBluescriptII SK+ (manufactured by StratageneCorporation) is preferable.

(Mutation of Amadoriase Gene)

Mutation of an amadoriase gene can be performed by any known methoddepending on an intended form of mutation. More specifically, a methodof bringing a chemical mutagen into contact with and allowing to act onan amadoriase gene or recombinant DNA comprising such gene integratedtherein; an ultraviolet irradiation method; a genetic engineeringtechnique; a method of making full use of a protein engineeringtechnique; or various other methods can be used.

Examples of chemical mutagens used in the aforementioned mutationinclude hydroxyl amine, N-methyl-N′-nitro-N-nitrosoguanidine, nitrousacid, sulfurous acid, hydrazine, formic acid, and 5-bromouracil.

Various conditions for the above contact/reactions may be employeddepending on the type of a drug to be used and are not particularlylimited where a desired mutation can be actually induced in anamadoriase gene. In general, the desired mutation can be induced bycontact/reactions performed at 20° C. to 80° C. for 10 minutes orlonger, and preferably 10 to 180 minutes, with the use of theaforementioned drug at the concentration of 0.5 M to 12 M. Theultraviolet irradiation may be also performed according to aconventional technique as described above (Gendai Kagaku, pp. 24-30,June, 1989).

To take advantage of protein engineering techniques, a technique knownas site-specific mutagenesis can in general be used. Examples includethe Kramer method (Nucleic Acids Res., 12, 9441, 1984; Methods Enzymol.,154, 350, 1987; Gene, 37, 73, 1985), the Eckstein method (Nucleic AcidsRes., 13, 8749, 1985; Nucleic Acids Res., 13, 8765, 1985; Nucleic AcidsRes, 14, 9679, 1986), and the Kunkel method (Proc. Natl. Acid. Sci.U.S.A., 82, 488, 1985; Methods Enzymol., 154, 367, 1987). As examples ofspecific methods to convert the base sequence within DNA, commerciallyavailable kits (Transformer Mutagenesis Kit, Clonetech; EXOIII/Mung BeanDeletion Kit, Stratagene; Quick Change Site Directed Mutagenesis Kit,Stratagene and the like) can be used.

A technique known as a general PCR (Polymerase Chain Reaction) techniquecan also be used (Technique, 1, 11, 1989). In addition to theconventional genetic mutation technique, by an organic synthesis methodor synthetic method of an enzyme, the modified amadoriase genes ofinterest can be also directly synthesized.

When determining or verifying the DNA nucleotide sequences of amadoriasegenes obtained by the aforementioned methods, the multi-capillary DNAanalysis system CEQ2000 (Beckman Coulter) and the like can, for example,be used.

(Transformation/transduction)

The amadoriase genes obtained as described above are integrated into avector such as a bacteriophage vector, a cosmid vector, or a plasmidvector used in transformation of a procaryotic or eucaryotic cell by aconventional technique, and a host corresponding to each vector can betransformed or transduced by a conventional technique. For example, theobtained recombinant DNA can be used on any microorganism, for examplemicroorganism belonging to the genus Escherichia, specifically E. coliK-12 strain, preferably E. coli JM109 strain or E. coli DH5aa strain(manufactured by Takara Bio Inc.) or an E. coli B strain, preferably E.coli BL21 strain (manufactured by Nippon gene Inc.) and the like totransform or transduce the same and to obtain the strain of interest.

(Identity of Amino Acid Sequences)

The identity of amino acid sequences can be obtained by calculationbased on a program such as maximum matching and search homology ofGENETYX Ver.11 (manufactured by GENETYX) or a program such as maximummatching and multiple alignment of DNASIS Pro (manufactured by HitachiSoftware).

(Determination of the Position Corresponding to Amino Acid)

A “position corresponding to an amino acid” refers to the positionpresent in the amino acid sequence of an amadoriase derived from otherspecies which corresponds to the amino acid at a particular position inthe amino acid sequence of the amadoriase derived from the genusConiochaeta as shown in SEQ ID No: 1.

A method of identifying the “position corresponding to an amino acid”may be performed by comparing amino acid sequences using a knownalgorithm such as the Lipman-Pearson method to assign maximum identityto conserved amino acid residues present in the amino acid sequence ofeach amadoriase. The positions of the homologous amino acid residues ineach of the amadoriase sequences can be determined, regardless ofinsertion or deletion of amino acid residue(s) in the amino acidsequences by aligning the amino acid sequences of the amadoriases bysuch method. Amino acid residues at homologous positions are thought toexist in similar positions in the three-dimensional structures, andamino acid residues at such homologous positions are expected to exertsimilar effects in terms of specificity of the amadoriase of interest.

FIGS. 1-1, 1-2 and 1-3 show the sequences of amadoriases derived fromvarious known species. The amino acid sequence as shown in SEQ ID No: 1is shown in the uppermost stage. The sequences shown in FIG. 1A-1C allhave an identity of 70% or higher with the sequence of SEQ ID No: 1 andwere aligned therewith based on a known algorithm. In the figures,mutation points within the variants of the present invention are shown.From FIGS. 1-1, 1-2, 1-3, one can recognize the positions in the aminoacid sequences of amadoriases derived from other species whichcorrespond to the amino acid at a particular position in the amino acidsequence of the amadoriase derived from the genus Coniochaeta. FIGS.1-1, 1-2 and 1-3 show the amino acid sequences of an amadoriase derivedfrom the genus Coniochaeta (SEQ ID No: 1), an amadoriase derived fromEupenicillium terrenum (SEQ ID No: 34), a ketoamine oxidase derived fromPyrenochaeta sp. (SEQ ID No: 35), a ketoamine oxidase derived fromArthrinium sp. (SEQ ID No: 36), a ketoamine oxidase derived fromCurvularia clavata (SEQ ID No: 37), a ketoamine oxidase derived fromNeocosmospora vasinfecta (SEQ ID No: 38), a fructosyl amino acid oxidasederived from Cryptococcus neoformans (SEQ ID No: 39), a fructosylpeptide oxidase derived from Phaeosphaeria nodorum (SEQ ID No: 40), afructosyl amino acid oxidase derived from Aspergillus nidulans (SEQ IDNo: 41), a fructosyl amino acid oxidase derived from Ulocladium sp. (SEQID No: 42), and a fructosyl amino acid oxidase derived from Penicilliumcrysogenum (SEQ ID No: 43).

(Position Corresponding to Substitution Site)

In the present invention, the phrase “the position corresponding toglutamic acid at position 44 in the amino acid sequence described in SEQID No: 1” refers to an amino acid corresponding to glutamic acid atposition 44 of an amadoriase of SEQ ID No: 1, when the identified aminoacid sequence of an amadoriase is compared with the amino acid sequenceof the amadoriase derived from the genus Coniochaeta and shown in SEQ IDNo: 1. Based on this, using the method of specifying the “amino acidresidue at the corresponding position”, the corresponding position canbe specified with reference to FIG. 1A in which amino acid sequences arealigned.

More specifically, the position corresponding to glutamic acid atposition 44 in the amino acid sequence described in SEQ ID No: 1 islysine at position 44 in the amadoriase derived from Eupenicilliumterrenum, proline at position 44 in the ketoamine oxidase derived fromPyrenochaeta sp., proline at position 44 in the ketoamine oxidasederived from Arthrinium sp., proline at position 44 in the ketoamineoxidase derived from Curvularia clavata, proline at position 44 in theketoamine oxidase derived from Neocosmospora vasinfecta, leucine atposition 44 in the fructosyl amino acid oxidase derived fromCryptococcus neoformans, proline at position 44 in the fructosyl peptideoxidase derived from Phaeosphaeria nodorum, proline at position 43 inthe fructosyl amino acid oxidase derived from Aspergillus nidulans,proline at position 44 in the fructosyl amino acid oxidase derived fromUlocladium sp., and proline at position 44 in the fructosyl amino acidoxidase derived from Penicillium crysogenum.

“The position corresponding to glutamic acid at position 81 in the aminoacid sequence described in SEQ ID No: 1” refers to an amino acidcorresponding to glutamic acid at position 81 of an amadoriase of SEQ IDNo: 1, when the identified amino acid sequence of an amadoriase iscompared with the amino acid sequence of the amadoriase derived from thegenus Coniochaeta and shown in SEQ ID No: 1. This can be also specifiedby the aforementioned method with reference to FIG. 1A in which aminoacid sequences are aligned.

More specifically, the position corresponding to glutamic acid atposition 81 in the amino acid sequence described in SEQ ID No: 1 isasparagine at position 81 in the amadoriase derived from Eupenicilliumterrenum, glutamic acid at position 81 in the ketoamine oxidase derivedfrom Pyrenochaeta sp., histidine at position 81 in the ketoamine oxidasederived from Arthrinium sp., glutamic acid at position 81 in theketoamine oxidase derived from Curvularia clavata, asparagine atposition 81 in the ketoamine oxidase derived from Neocosmosporavasinfecta, asparagine at position 81 in the fructosyl amino acidoxidase derived from Cryptococcus neoformans, glutamic acid at position81 in the fructosyl peptide oxidase derived from Phaeosphaeria nodorum,asparagine at position 80 in the fructosyl amino acid oxidase derivedfrom Aspergillus nidulans, glutamic acid at position 81 in the fructosylamino acid oxidase derived from Ulocladium sp., and asparagine atposition 81 in the fructosyl amino acid oxidase derived from Penicilliumcrysogenum.

“The position corresponding to glutamic acid at position 133 in theamino acid sequence described in SEQ ID No: 1” refers to an amino acidcorresponding to glutamic acid at position 133 of the amino acidsequence described in SEQ ID No: 1, when the identified amino acidsequence of an amadoriase is compared with the amino acid sequence ofthe amadoriase derived from the genus Coniochaeta and shown in SEQ IDNo: 1. This can be specified based on FIG. 1A, in which amino acidsequences are aligned by the aforementioned method.

More specifically, the position corresponding to glutamic acid atposition 133 in the amino acid sequence described in SEQ ID No: 1 isglutamic acid at position 133 in the amadoriase derived fromEupenicillium terrenum, glutamic acid at position 133 in the ketoamineoxidase derived from Pyrenochaeta sp., alanine at position 133 in theketoamine oxidase derived from Arthrinium sp., glutamic acid at position133 in the ketoamine oxidase derived from Curvularia clavata, alanine atposition 133 in the ketoamine oxidase derived from Neocosmosporavasinfecta, glutamic acid at position 133 in the fructosyl amino acidoxidase derived from Cryptococcus neoformans, glutamic acid at position131 in the fructosyl peptide oxidase derived from Phaeosphaeria nodorum,glutamic acid at position 132 in the fructosyl amino acid oxidasederived from Aspergillus nidulans, lysine at position 133 in thefructosyl amino acid oxidase derived from Ulocladium sp., and asparticacid at position 133 in the fructosyl amino acid oxidase derived fromPenicillium crysogenum.

“The position corresponding to glutamic acid at position 253 in theamino acid sequence described in SEQ ID No: 1” refers to an amino acidcorresponding to glutamic acid at position 253 of amino acid sequence ofSEQ ID No: 1, when the identified amino acid sequence of an amadoriaseis compared with the amino acid sequence of the amadoriase derived fromthe genus Coniochaeta and shown in SEQ ID No: 1. This can be alsospecified by the aforementioned method with reference to FIG. 1B inwhich amino acid sequences are aligned.

More specifically, the position corresponding to glutamic acid atposition 253 in the amino acid sequence described in SEQ ID No: 1 isalanine at position 253 in the amadoriase derived from Eupenicilliumterrenum, alanine at position 251 in the ketoamine oxidase derived fromPyrenochaeta sp., glutamic acid at position 253 in the ketoamine oxidasederived from Arthrinium sp., glutamic acid at position 251 in theketoamine oxidase derived from Curvularia clavata, valine at position253 in the ketoamine oxidase derived from Neocosmospora vasinfecta,glutamic acid at position 253 in the fructosyl amino acid oxidasederived from Cryptococcus neoformans, arginine at position 249 in thefructosyl peptide oxidase derived from Phaeosphaeria nodorum, alanine atposition 253 in the fructosyl amino acid oxidase derived fromAspergillus nidulans, glutamic acid at position 251 in the fructosylamino acid oxidase derived from Ulocladium sp., and glutamine atposition 253 in the fructosyl amino acid oxidase derived fromPenicillium crysogenum.

“The position corresponding to glycine at position 256 in the amino acidsequence described in SEQ ID No: 1” refers to an amino acidcorresponding to glycine at position 256 of an amadoriase of SEQ ID No:1, when the identified amino acid sequence of an amadoriase is comparedwith the amino acid sequence of the amadoriase derived from the genusConiochaeta and shown in SEQ ID No: 1. This can be also specified by theaforementioned method with reference to FIG. 1B in which amino acidsequences are aligned.

More specifically, the position corresponding to glycine at position 256in the amino acid sequence described in SEQ ID No: 1 is asparagine atposition 256 in the amadoriase derived from Eupenicillium terrenum,aspartic acid at position 254 in the ketoamine oxidase derived fromPyrenochaeta sp., glycine at position 256 in the ketoamine oxidasederived from Arthrinium sp., asparagine at position 254 in the ketoamineoxidase derived from Curvularia clavata, glycine at position 256 in theketoamine oxidase derived from Neocosmospora vasinfecta, glutamic acidat position 256 in the fructosyl amino acid oxidase derived fromCryptococcus neoformans, asparagine at position 252 in the fructosylpeptide oxidase derived from Phaeosphaeria nodorum, asparagine atposition 256 in the fructosyl amino acid oxidase derived fromAspergillus nidulans, asparagine at position 254 in the fructosyl aminoacid oxidase derived from Ulocladium sp., and aspartic acid at position256 in the fructosyl amino acid oxidase derived from Penicilliumcrysogenum.

“The position corresponding to valine at position 257 in the amino acidsequence described in SEQ ID No: 1” refers to an amino acidcorresponding to valine at position 257 of an amadoriase of SEQ ID No:1, when the identified amino acid sequence of an amadoriase is comparedwith the amino acid sequence of the amadoriase derived from the genusConiochaeta and shown in SEQ ID No: 1. This can be also specified by theaforementioned method with reference to FIG. 1B in which amino acidsequences are aligned.

More specifically, the position corresponding to valine at position 257in the amino acid sequence described in SEQ ID No: 1 is valine atposition 257 in the amadoriase derived from Eupenicillium terrenum,threonine at position 255 in the ketoamine oxidase derived fromPyrenochaeta sp., cysteine at position 257 in the ketoamine oxidasederived from Arthrinium sp., valine at position 255 in the ketoamineoxidase derived from Curvularia clavata, cysteine at position 257 in theketoamine oxidase derived from Neocosmospora vasinfecta, cysteine atposition 257 in the fructosyl amino acid oxidase derived fromCryptococcus neoformans, serine at position 253 in the fructosyl peptideoxidase derived from Phaeosphaeria nodorum, threonine at position 257 inthe fructosyl amino acid oxidase derived from Aspergillus nidulans,valine at position 255 in the fructosyl amino acid oxidase derived fromUlocladium sp., and valine at position 257 in the fructosyl amino acidoxidase derived from Penicillium crysogenum.

“The position corresponding to asparagine at position 262 in the aminoacid sequence described in SEQ ID No: 1” refers to an amino acidcorresponding to asparagine at position 262 of an amadoriase of SEQ IDNo: 1, when the identified amino acid sequence of an amadoriase iscompared with the amino acid sequence of the amadoriase derived from thegenus Coniochaeta and shown in SEQ ID No: 1. This can be also specifiedby the aforementioned method with reference to FIG. 1B in which aminoacid sequences are aligned.

More specifically, the position corresponding to asparagine at position262 in the amino acid sequence described in SEQ ID No: 1 is asparticacid at position 262 in the amadoriase derived from Eupenicilliumterrenum, asparagine at position 260 in the ketoamine oxidase derivedfrom Pyrenochaeta sp., histidine at position 262 in the ketoamineoxidase derived from Arthrinium sp., asparagine at position 260 in theketoamine oxidase derived from Curvularia clavata, histidine at position262 in the ketoamine oxidase derived from Neocosmospora vasinfecta,asparagine at position 262 in the fructosyl amino acid oxidase derivedfrom Cryptococcus neoformans, asparagine at position 258 in thefructosyl peptide oxidase derived from Phaeosphaeria nodorum, asparticacid at position 262 in the fructosyl amino acid oxidase derived fromAspergillus nidulans, asparagine at position 260 in the fructosyl aminoacid oxidase derived from Ulocladium sp., and aspartic acid at position262 in the fructosyl amino acid oxidase derived from Penicilliumcrysogenum.

“The position corresponding to glutamine at position 337 in the aminoacid sequence described in SEQ ID No: 1” refers to an amino acidcorresponding to glutamine at position 337 of an amadoriase of SEQ IDNo: 1, when the identified amino acid sequence of an amadoriase iscompared with the amino acid sequence of the amadoriase derived from thegenus Coniochaeta and shown in SEQ ID No: 1. This can be also specifiedby the aforementioned method with reference to FIG. 1B in which aminoacid sequences are aligned.

More specifically, the position corresponding to glutamine at position337 in the amino acid sequence described in SEQ ID No: 1 is lysine atposition 337 in the amadoriase derived from Eupenicillium terrenum,lysine at position 335 in the ketoamine oxidase derived fromPyrenochaeta sp., glutamine at position 338 in the ketoamine oxidasederived from Arthrinium sp., threonine at position 335 in the ketoamineoxidase derived from Curvularia clavata, lysine at position 337 in theketoamine oxidase derived from Neocosmospora vasinfecta, lysine atposition 337 in the fructosyl amino acid oxidase derived fromCryptococcus neoformans, lysine at position 333 in the fructosyl peptideoxidase derived from Phaeosphaeria nodorum, asparagine at position 337in the fructosyl amino acid oxidase derived from Aspergillus nidulans,threonine at position 335 in the fructosyl amino acid oxidase derivedfrom Ulocladium sp., and lysine at position 337 in the fructosyl aminoacid oxidase derived from Penicillium crysogenum.

“The position corresponding to glutamic acid at position 340 in theamino acid sequence described in SEQ ID No: 1” refers to an amino acidcorresponding to glutamic acid at position 340 of an amadoriase of SEQID No: 1, when the identified amino acid sequence of an amadoriase iscompared with the amino acid sequence of the amadoriase derived from thegenus Coniochaeta and shown in SEQ ID No: 1. This can be also specifiedby the aforementioned method with reference to FIG. 1B in which aminoacid sequences are aligned.

More specifically, the position corresponding to glutamic acid atposition 340 in the amino acid sequence described in SEQ ID No: 1 isglutamic acid at position 340 in the amadoriase derived fromEupenicillium terrenum, glutamic acid at position 338 in the ketoamineoxidase derived from Pyrenochaeta sp., glutamic acid at position 341 inthe ketoamine oxidase derived from Arthrinium sp., glutamic acid atposition 338 in the ketoamine oxidase derived from Curvularia clavata,proline at position 340 in the ketoamine oxidase derived fromNeocosmospora vasinfecta, glutamic acid at position 340 in the fructosylamino acid oxidase derived from Cryptococcus neoformans, lysine atposition 336 in the fructosyl peptide oxidase derived from Phaeosphaerianodorum, glutamic acid at position 340 in the fructosyl amino acidoxidase derived from Aspergillus nidulans, glutamic acid at position 338in the fructosyl amino acid oxidase derived from Ulocladium sp., andglutamic acid at position 340 in the fructosyl amino acid oxidasederived from Penicillium crysogenum.

“The position corresponding to aspartic acid at position 129 in theamino acid sequence described in SEQ ID No: 1” refers to an amino acidcorresponding to aspartic acid at position 129 of an amadoriase of SEQID No: 1, when the identified amino acid sequence of an amadoriase iscompared with the amino acid sequence of the amadoriase derived from thegenus Coniochaeta and shown in SEQ ID No: 1. This can be also specifiedby the aforementioned method with reference to FIG. 1A in which aminoacid sequences are aligned.

More specifically, the position corresponding to aspartic acid atposition 129 in the amino acid sequence described in SEQ ID No: 1 isglutamic acid at position 129 in the amadoriase derived fromEupenicillium terrenum, aspartic acid at position 129 in the ketoamineoxidase derived from Pyrenochaeta sp., aspartic acid at position 129 inthe ketoamine oxidase derived from Arthrinium sp., aspartic acid atposition 129 in the ketoamine oxidase derived from Curvularia clavata,aspartic acid at position 129 in the ketoamine oxidase derived fromNeocosmospora vasinfecta, serine at position 129 in the fructosyl aminoacid oxidase derived from Cryptococcus neoformans, aspartic acid atposition 127 in the fructosyl peptide oxidase derived from Phaeosphaerianodorum, glutamic acid at position 128 in the fructosyl amino acidoxidase derived from Aspergillus nidulans, aspartic acid at position 129in the fructosyl amino acid oxidase derived from Ulocladium sp., andglutamic acid at position 129 in the fructosyl amino acid oxidasederived from Penicillium crysogenum.

“The position corresponding to aspartic acid at position 132 in theamino acid sequence described in SEQ ID No: 1” refers to an amino acidcorresponding to aspartic acid at position 132 of an amadoriase of SEQID No: 1, when the identified amino acid sequence of an amadoriase iscompared with the amino acid sequence of the amadoriase derived from thegenus Coniochaeta and shown in SEQ ID No: 1. This can be also specifiedby the aforementioned method with reference to FIG. 1A in which aminoacid sequences are aligned.

More specifically, the position corresponding to aspartic acid atposition 132 in the amino acid sequence described in SEQ ID No: 1 isaspartic acid at position 132 in the amadoriase derived fromEupenicillium terrenum, aspartic acid at position 132 in the ketoamineoxidase derived from Pyrenochaeta sp., aspartic acid at position 132 inthe ketoamine oxidase derived from Arthrinium sp., aspartic acid atposition 132 in the ketoamine oxidase derived from Curvularia clavata,glutamic acid at position 132 in the ketoamine oxidase derived fromNeocosmospora vasinfecta, aspartic acid at position 132 in the fructosylamino acid oxidase derived from Cryptococcus neoformans, aspartic acidat position 130 in the fructosyl peptide oxidase derived fromPhaeosphaeria nodorum, aspartic acid at position 131 in the fructosylamino acid oxidase derived from Aspergillus nidulans, aspartic acid atposition 132 in the fructosyl amino acid oxidase derived from Ulocladiumsp., and aspartic acid at position 132 in the fructosyl amino acidoxidase derived from Penicillium crysogenum.

“The position corresponding to glutamic acid at position 231 in theamino acid sequence described in SEQ ID No: 1” refers to an amino acidcorresponding to glutamic acid at position 231 of an amadoriase of SEQID No: 1, when the identified amino acid sequence of an amadoriase iscompared with the amino acid sequence of the amadoriase derived from thegenus Coniochaeta and shown in SEQ ID No: 1. This can be also specifiedby the aforementioned method with reference to FIG. 1B in which aminoacid sequences are aligned.

More specifically, the position corresponding to glutamic acid atposition 231 in the amino acid sequence described in SEQ ID No: 1 isglutamic acid at position 231 in the amadoriase derived fromEupenicillium terrenum, glutamic acid at position 229 in the ketoamineoxidase derived from Pyrenochaeta sp., glutamic acid at position 231 inthe ketoamine oxidase derived from Arthrinium sp., glutamic acid atposition 229 in the ketoamine oxidase derived from Curvularia clavata,glutamic acid at position 231 in the ketoamine oxidase derived fromNeocosmospora vasinfecta, glutamic acid at position 231 in the fructosylamino acid oxidase derived from Cryptococcus neoformans, histidine atposition 227 in the fructosyl peptide oxidase derived from Phaeosphaerianodorum, glutamic acid at position 231 in the fructosyl amino acidoxidase derived from Aspergillus nidulans, glutamine at position 229 inthe fructosyl amino acid oxidase derived from Ulocladium sp., andglutamic acid at position 231 in the fructosyl amino acid oxidasederived from Penicillium crysogenum.

“The position corresponding to aspartic acid at position 232 in theamino acid sequence described in SEQ ID No: 1” refers to an amino acidcorresponding to aspartic acid at position 232 of an amadoriase of SEQID No: 1, when the identified amino acid sequence of an amadoriase iscompared with the amino acid sequence of the amadoriase derived from thegenus Coniochaeta and shown in SEQ ID No: 1. This can be also specifiedby the aforementioned method with reference to FIG. 1B in which aminoacid sequences are aligned.

More specifically, the position corresponding to aspartic acid atposition 232 in the amino acid sequence described in SEQ ID No: 1 isaspartic acid at position 232 in the amadoriase derived fromEupenicillium terrenum, aspartic acid at position 230 in the ketoamineoxidase derived from Pyrenochaeta sp., glutamic acid at position 232 inthe ketoamine oxidase derived from Arthrinium sp., aspartic acid atposition 230 in the ketoamine oxidase derived from Curvularia clavata,glutamic acid at position 232 in the ketoamine oxidase derived fromNeocosmospora vasinfecta, glycine at position 232 in the fructosyl aminoacid oxidase derived from Cryptococcus neoformans, glutamic acid atposition 228 in the fructosyl peptide oxidase derived from Phaeosphaerianodorum, glutamic acid at position 232 in the fructosyl amino acidoxidase derived from Aspergillus nidulans, aspartic acid at position 230in the fructosyl amino acid oxidase derived from Ulocladium sp., andaspartic acid at position 232 in the fructosyl amino acid oxidasederived from Penicillium crysogenum.

“The position corresponding to glutamic acid at position 249 in theamino acid sequence described in SEQ ID No: 1” refers to an amino acidcorresponding to glutamic acid at position 249 of an amadoriase of SEQID No: 1, when the identified amino acid sequence of an amadoriase iscompared with the amino acid sequence of the amadoriase derived from thegenus Coniochaeta and shown in SEQ ID No: 1. This can be also specifiedby the aforementioned method with reference to FIG. 1B in which aminoacid sequences are aligned.

More specifically, the position corresponding to glutamic acid atposition 249 in the amino acid sequence described in SEQ ID No: 1 islysine at position 249 in the amadoriase derived from Eupenicilliumterrenum, lysine of position 247 in the ketoamine oxidase derived fromPyrenochaeta sp., histidine at position 249 in the ketoamine oxidasederived from Arthrinium sp., glutamic acid at position 247 in theketoamine oxidase derived from Curvularia clavata, glutamic acid atposition 249 in the ketoamine oxidase derived from Neocosmosporavasinfecta, glutamic acid at position 249 in the fructosyl amino acidoxidase derived from Cryptococcus neoformans, glutamic acid at position245 in the fructosyl peptide oxidase derived from Phaeosphaeria nodorum,alanine at position 249 in the fructosyl amino acid oxidase derived fromAspergillus nidulans, serine at position 247 in the fructosyl amino acidoxidase derived from Ulocladium sp., and glutamine at position 249 inthe fructosyl amino acid oxidase derived from Penicillium crysogenum.

(Production of the Amadoriase of the Present Invention)

In order to use a strain having the ability to produce an amadoriasehaving excellent resistance to detergents obtained as described aboveand produce said amadoriase, the strain may be cultured by aconventional solid culture method, although liquid culture is preferablewhere possible.

Examples of media to culture the aforementioned strains include mediaprepared by adding one or more inorganic salts, such as sodium chloride,monopotassium phosphate, dipotassium phosphate, magnesium sulfate,magnesium chloride, ferric chloride, ferric sulfate, and manganesesulfate, to one or more nitrogen sources, such as a yeast extract,tryptone, peptone, a meat extract, a corn steep liquor, and a leachingsolution of soybean or wheat bran, and further adding saccharinematerials, vitamins, and the like thereto, where necessary.

Incidentally, it is appropriate to adjust the initial pH of the media asfrom 7 to 9.

Further, culture can be performed under any condition and, for example,culture can be performed at 20° C. to 42° C., and more preferably atabout 30° C. for 4 to 24 hours, and further preferably at about 30° C.for 8 to 16 hours, by, for example, aeration spinner submerged culture,shake culture, or stationary culture.

Following the completion of culture, amadoriases may be collected fromthe culture products with conventional enzyme collecting means. Forexample, a strain may be subjected to ultrasonic disintegrationtreatment or grinding treatment by a conventional method, the enzyme maybe extracted using a lytic enzyme such as lysozyme, or bacteriolysis maybe performed via shaking or still standing in the presence of toluene toexcrete the enzyme from the microorganism body. The solution is filteredor centrifuged to remove solid content, and according to need, nucleicacid is removed with the aid of streptomycin sulfate, protamine sulfate,or manganese sulfate, and to this ammonium sulfate, alcohol, or acetoneis added to the solution so as to fractionate the solution, andsediments are then collected to obtain the crude enzymes of theamadoriases.

The purified amadoriase enzyme preparation can be obtained from: thecrude enzyme of the aforementioned amadoriase by a method appropriatelyselected from gel filtration methods using Sephadex, Superdex, orUltrogel; adsorption-elution methods using ion exchange carriers;electrophoretic methods using polyacrylamide gels, etc.;adsorption-elution methods using hydroxyapatite; sedimentation methodssuch as sucrose density-gradient centrifugation; affinitychromatographic methods; and fractionation methods using a molecularsieve membrane, a hollow-fiber membrane, etc. Alternatively, theaforementioned methods can adequately be performed in combination. Theamadoriase having improved substrate specificity of interest can thus beobtained.

(Surfactant of the Present Invention)

The surfactant of the present invention is not particularly limited solong as a method of measuring HbAlc of the present invention can becarried out in the presence of the surfactant, and a nonionic surfactantand an ionic surfactant such as a cationic surfactant, an anionicsurfactant and an amphoteric surfactant, can be mentioned andparticularly, a cationic surfactant and an anionic surfactant arepreferable. The expression surfactant when mentioned in the presentspecification encompasses one or more surfactants unless otherwiseindicated.

The surfactant of the present invention can be a surfactant having acritical micelle concentration (CMC) of 70 mM or lower, 50 mM or lower,20 mM or lower, 10 mM or lower, 7 mM or lower, 6 mM or lower, 5 mM orlower, 4.5 mM or lower, 4 mM or lower, 3.5 mM or lower, 3 mM or lower,2.5 mM or lower, 2 mM or lower, 1.5 mM or lower, 1.3 mM or lower, or 1mM or lower. In an embodiment, the critical micelle concentration of thesurfactant of the present invention can be 0.1 mM or 0.01 mM or higher,preferably 50 mM or lower, more preferably 20 mM or lower, morepreferably 10 mM or lower, more preferably 7 mM or lower, morepreferably 6 mM or lower, and most preferably 5 mM or lower. Thecritical micelle concentration refers to the critical concentrationabove which micelles of a surfactant are formed in a solution and belowwhich micelles are not formed. In general, the lower the criticalmicelle concentration, the lower the concentration of a surfactantforming micelles and the stronger the surfactant action. A personskilled in the art can determine the critical micelle concentration of adesired surfactant by conventional methods. For example, a commerciallyavailable kit, which measures a critical micelle concentration of asurfactant based on a change in fluorescence of a fluorescent reagentinteracting with the surfactant, can be used (for example, DetergentCritical Micelle Concentration (CMC) Assay Kit manufactured by PFPInc.).

For example, the CMC of octyltrimethylammonium bromide (C8, OTAB) isabout 140 mM; the CMC of decyltrimethylammonium chloride (C10) is about65 mM; the CMC of decyltrimethylammonium bromide (C10) is about 70 mM;the CMC of dodecyltrimethylammonium chloride (C12) is about 20 mM; theCMC of dodecyltrimethylammonium bromide (C12, DTAB) is about 16 mM; theCMC of tetradecyltrimethylammonium chloride (C14, TTAC) is about 4.5 mM;the CMC of tetradecyltrimethylammonium bromide (C14, TTAB) is about 5mM; the CMC of hexadecyltrimethylammonium chloride (C16, CTAC) is about1.3 mM; the CMC of hexadecyltrimethylammonium bromide (C16) is about 1mM; the CMC of octadecyltrimethylammonium chloride (C18, STAC) is about0.3 mM; and the CMC of octadecyltrimethylammonium bromide (C18, STAB) isabout 0.3 mM (for example, see J. PHYS. COLLIDE. CHEM., 52, 130 (1948);J. PHYS. CHEM., 66, 1839 (1962); J. AM. OIL. CHEMISTS. SOC., 30, 74(1953); J. PHARM. SCI., 54, 436 (1965); KONINKI. NED. AKAD. WETEN. PROC.SER B, 58, 97 (1955); J. PHYS. CHEM., 65, 1807 (1961); J. AM. CHEM.SOC., 65, 692 (1943); J. AM. CHEM. SOC., 69, 2095 (1947); J. COLLIDE.INTERFACE. SCI., 22, 430 (1966); and J. AM. CHEM. SOC., 70, 3803(1948)). The numerals within parentheses indicate the number of carbonatoms of the longest substituent.

For example, the CMC of 1-dodecylpyridinium bromide (C12) is about 12mM; the CMC of 1-dodecylpyridinium chloride (C12, 1-DPC) is about 14 mM;the CMC of 1-tetradecylpyridinium bromide (C14) is about 2.9 mM; the CMCof 1-hexadecylpyridinium chloride (C16, 1-CPC) is about 0.6 mM; the CMCof 1-hexadecylpyridinium bromide (C16, 1-CPB) is about 0.9 mM; the CMCof N-cetyl-4-methylpyridinium chloride (C16, 4Me-1-CPC) is about 1.9 mM;the CMC of 1-octadecylpyridinium bromide is about 0.6 mM; and the CMC of1-octadecylpyridinium chloride is about 0.24 mM (see, for example, J.COLLIDE. INTERFACE. SCI., 21, 522 (1966); J. PHARM. SCI., 54, 436(1965); TRANS. FARADAY. SOC., 62, 3244 (1966); J. AM. CHEM. SOC., 70,3803 (1948); REV. CHIM. AC. REP. POP. ROUM., 6, 309 (1961); and J. AM.CHEM. SOC., 70, 3049 (1948)).

The CMC of benzyldodecyldimethylammonium chloride is about 2.8 mM; theCMC of benzyltetradecyldimethylammonium chloride (C14, BDTAC) is about0.37 mM; and the CMC of benzylcetyldimethylammonium chloride (C16,BCDAC) is about 0.042 mM (see, for example, surfactant handbook, 131(1960), J. COLLIDE. INTERFACE. SCI., 22, 430 (1966); and J. COLLIDE.SCI., 8, 385 (1953)).

Examples of the non-ionic surfactant include a polyoxyethylene alkylether, a sorbitan fatty acid ester, an alkyl polyglucoside, a fatty aciddiethanol amide and an alkyl monoglyceryl ether.

Examples of the cationic surfactant include an alkyltrimethylammoniumsalt, a dialkyldimethylammonium salt, an alkylbenzyldimethyl ammoniumsalt, a pyridinium salt such as an alkylpyridinium salt, a phosphoniumsalt such as an alkylphosphonium salt, an imidazolium salt such as analkylimidazolium salt, and an isoquinolinium salt such as analkylisoquinolinium salt.

Examples of the cationic surfactant of the present invention include aquaternary ammonium salt (I), a pyridinium salt (II) and a phosphoniumsalt (III) represented by the following general formulae.

[wherein, R¹ to R⁴, which may be the same or different, each represent asubstituted or unsubstituted C₁ to C₂₀ alkyl, alkenyl, aryl or benzyl;and Z⁻ represents a monovalent anion].

[wherein, R⁵ represents a substituted or unsubstituted C₁ to C₂₀ alkyl;each R^(a), which may be the same or different, represents a hydrogenatom or a substituted or unsubstituted C₁ to C₂₀ alkyl, alkenyl, aryl orbenzyl; n represents an integer of 1 to 5 and Z⁻ represents a monovalentanion].

[wherein, R⁶ to R⁹, which may be the same or different, each represent asubstituted or unsubstituted C₁ to C₂₀ alkyl, alkenyl, aryl or benzyl;and Z⁻ represents a monovalent anion].

Examples of the quaternary ammonium salt include octyltrimethylammoniumchloride (OTAC), octyltrimethylammonium bromide (OTAB),decyltrimethylammonium chloride, decyltrimethylammonium bromide (DTAB),dodecyltrimethylammonium chloride, dodecyltrimethylammonium bromide,tetradecyltrimethylammonium chloride (TTAC), tetradecyltrimethylammoniumbromide (TTAB), hexadecyltrimethylammonium chloride (CTAC),hexadecyltrimethylammonium bromide, octadecyltrimethylammonium chloride,octadecyltrimethylammonium bromide (STAB), eicosyltrimethylammoniumchloride, eicosyltrimethylammonium bromide,benzyldodecyldimethylammonium chloride, benzyldodecyldimethylammoniumbromide (BDDAB), benzyltetradecyldimethylammonium chloride (BDTAC),benzyltetradecyldimethyl ammonium bromide, benzylcetyldimethyl ammoniumchloride (BCDAC), benzylcetyldimethylammonium bromide,dioctyldimethylammonium chloride and dioctyldimethylammonium bromide.

Examples of the pyridinium salt include 1-decylpyridinium chloride,1-decylpyridinium bromide, 1-dodecylpyridinium chloride (1-DPC),1-dodecylpyridinium bromide, 1-tetradecylpyridinium chloride,1-tetradecylpyridinium bromide, 1-hexadecylpyridinium chloride (1-CPC),1-hexadecylpyridinium bromide (1-CPB), N-cetyl-2-methylpyridiniumchloride, N-cetyl-3-methylpyridinium chloride,N-cetyl-4-methylpyridinium chloride (4Me-1-CPC), 1-octadecylpyridiniumchloride, 1-octadecylpyridinium bromide, 1-eicosylpyridinium chlorideand 1-eicosylpyridinium bromide.

Examples of the phosphonium salt include tetraethylphosphonium chloride,tetraethylphosphonium bromide, tributylmethylphosphonium chloride,tributylmethylphosphonium bromide, tributylmethylphosphonium iodide,tetrabutylphosphonium chloride, tetrabutylphosphonium bromide,tetra-n-octylphosphonium chloride, tetra-n-octylphosphonium bromide,tributyldodecylphosphonium chloride, tributyldodecylphosphonium bromide,tributylhexadecylphosphonium chloride, tributylhexadecylphosphoniumbromide (TBCPB), methyltriphenylphosphonium chloride,methyltriphenylphosphonium bromide, methyltriphenylphosphonium iodide,tetraphenylphosphonium chloride and tetratetraphenylphosphonium bromide.

Anion Z⁻ to be pair up with a cationic surfactant, can, for example, beCl⁻, Br⁻ or I⁻.

Examples of the anionic surfactant include a linear alkylbenzenesulfonate, an alkyl sulfate, an alpha-olefin sulfonate, apolyoxyethylene alkyl ether sulfate, an ca-sulfo fatty acid ester saltand an alkali metal salt of a natural fatty acid. Examples of such asurfactant include sodium dodecyl sulfate (SDS).

Examples of the amphoteric surfactant include an alkyl dimethyl amineoxide and alkylcarboxybetaine.

(Kit Containing an Amadoriase and Surfactant of the Present Invention)

The present invention provides a kit for measuring glycated hemoglobin,containing an amadoriase and a surfactant. The surfactant can be anonionic or ionic surfactant. The amadoriase and the surfactant can becontained as a mixture or discrete components. When an amadoriase and asurfactant are contained as a mixture in a kit, it is generallypreferable that the surfactant is contained at a concentration at whichthe amadoriase is not inactivated. When the amadoriase and surfactantare contained as discrete components in the kit, a stock solutioncontaining a surfactant at a higher concentration than the finalconcentration used for measurement may be used as the surfactant. Thisstock solution is appropriately diluted to prepare the solution for usedin measurement.

The kit containing an amadoriase and a surfactant of the presentinvention can further contain a reagent for measuring αFVH, a proteaseor peptidase for cleaving αFVH and other components, i.e., a stabilizerand a buffer solution known in the art. Techniques used in kits formeasuring αFVH can be appropriately used for producing a kit containingan amadoriase of the present invention and a surfactant. Morespecifically, the present invention provides a method for producing akit containing an amadoriase and a surfactant comprising the step ofpreparing an appropriate amadoriase and surfactant. In this case, theamadoriase and surfactant can be prepared as a mixture or discretecomponents. When the amadoriase and surfactant are provided as discretecomponents in a kit, they can be mixed immediately before measurement ofαFVH.

The amadoriase contained in the kit of the present invention preferablyexhibits a residual activity (%) of preferably 13% or higher, morepreferably 15% or higher, most preferably 19% or higher, (for example,20% or higher, 30% or higher, 40% or higher, 50% or higher, 60% orhigher, 70% or higher, 80% or higher, 90% or higher, 95% or higher or99% or higher) 5 minutes after a surfactant solution controlled to havea final concentration is added, compared with the amadoriase to whichthe surfactant solution is not added. The residual activity will bedescribed below.

The amadoriase contained in the kit of the present invention has a finalconcentration of 110 μg/ml or lower (for example, 100 μg/ml or lower, 70μg/ml or lower, or 50 μg/ml or lower) per surfactant of 0.01% (w/v)preferably at the time of measurement. The surfactant contained in thekit has a final concentration at the time of measurement of 0.01% (w/v)or higher (for example, 0.02% (w/v) or higher, 0.04% (w/v) or higher,0.05% (w/v) or higher, 0.06% (w/v) or higher, 0.07% (w/v) or higher,0.08% (w/v) or higher, 0.09% (w/v) or higher, 0.1% (w/v) or higher,0.15% (w/v) or higher, 0.2% (w/v) or higher, 0.25% (w/v) or higher, or0.3% (w/v) or higher). The final concentration at the time ofmeasurement herein refers to the concentration of the component finallydiluted and used for measuring glycated hemoglobin. Accordingly, the kitmay contain a stock solution having a higher concentration than thefinal concentration at the time of measurement.

The amadoriase contained in the kit of the present invention can be anamadoriase having the amino acid sequence as shown in SEQ ID No: 1 orSEQ ID NO: 37 or a variant prepared based on the same with improvedsurfactant resistance. The variant may have an amino acid sequencehaving an sequence identity of, for example, 70% or higher, 75% orhigher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 97%or higher, or 99% or higher, with SEQ ID No: 1 or SEQ ID No: 37 or anamino acid sequence prepared by modifying or altering one or severalamino acids in the amino acid sequence as shown in SEQ ID No: 1 or SEQID No: 37 or deleting, substituting, adding and/or inserting a single toseveral amino acids in the amino acid sequence.

The amadoriase contained in the kit of the present invention can be anaturally occurring amadoriase derived from the genara Eupenicillium,Pyrenochaeta, Arthrinium, Curvularia, Neocosmospora, Cryptococcus,Phaeosphaeria, Aspergillus, Emericella, Ulocladium, Penicillium,Fusarium, Achaetomiella, Achaetomium, Thielavia, Chaetomium,Gelasinospora, Microascus, Leptosphaeria, Ophiobolus, Pleospora,Coniochaetidium, Pichia, Corynebacterium, Agrobacterium and Arthrobacteror a variant thereof. Such variant may have one or more amino-acidsubstitutions at the position corresponding to an amino acid selectedfrom the group consisting of asparagine at position 262, valine atposition 257, glutamic acid at position 253, glutamine at position 337,glutamic acid at position 340, glutamic acid at position 133, glutamicacid at position 44, glycine at position 256, glutamic acid at position81, aspartic acid at aspartic acid at position 129 at position 132,glutamic acid at position 231, aspartic acid at position 232 andglutamic acid at position 249 in the amino acid sequence as shown in SEQID NO: 1 or 3. A person skilled in the art can readily confirm whetheror not an amadoriase or a variant thereof can be used in the kit of thepresent invention, more specifically, whether or not an amadoriase hasdesired surfactant resistance, by e.g., using the test method describedlater or the evaluation method in Example 7.

(Buffer)

To the kit or composition of the present invention, a buffer or a buffersolution having a buffer capacity within the range of pH5.0 to pH10.0,preferably pH6.0 to pH8.0 which is a range in which amadoriase is notinactivated, can appropriately be added. The term buffer as mentioned inthe present specification is defined to include one or more buffersunless otherwise indicated. The term buffer solution refers to asolution having a buffer action (buffer capacity) of maintaining the pHof a solution within a constant range; whereas the term buffer (bufferagent) refers to an agent which confers buffer action to a solution. Abuffer, if a weak acid is taken as an example, is composed of a weakacid and a salt thereof. In this case, the salt is referred to as aconjugate salt. For example, if a buffer is composed of a phosphoricacid and a potassium salt thereof, since a base compound is a phosphoricacid, such a buffer is sometimes referred to as a phosphate buffer inthe present specification for convenience. The concentration of a bufferrefers to the concentration of the base compound, which is a total ofthe compound alone serving as a base of the buffer and the conjugatesalt form thereof. For example, the expression 100 mM of phosphatebuffer means the total phosphoric-acid concentration, which is a totalof the phosphoric acid and conjugate salt thereof (for example,potassium phosphate) contained in the solution at final concentration,is 100 mM.

Among buffers (buffer solutions), in particular, those which maintainthe residual activity of an amadoriase in the presence of a surfactantor which alleviate reduction of residual activity, are preferable. Inthe present specification, such preferable buffer may be particularlyreferred to as a buffer having an amadoriase stabilizing effect or thebuffer of the present invention. For example, HEPES does not have anamadoriase stabilizing effect on an amadoriase derived from the genusConiochaeta (CFP-T7, SEQ ID No: 1), even if it is used in aconcentration of 500 mM (pH 7.0). Thus, HEPES does not fall under abuffer having an amadoriase stabilizing effect of the present invention.As can be seen, not all buffers have amadoriase stabilizing effects.Thus, the buffer having the amadoriase stabilizing effect of the presentinvention not only maintains the pH of a solution at a constant levelbut also has the effect of stabilizing an amadoriase in buffering pH.The amadoriase stabilizing effect of the buffer of the present inventionherein refers to an action of maintaining the residual activity of anamadoriase in the presence of a surfactant, or an action of alleviatingreduction of residual activity. Such amadoriase stabilizing effect(action) can be evaluated by comparing the residual amadoriase activityof a solution which does not contain any buffer or a solution using abuffer which does not have amadoriase stabilizing effect of the presentinvention with the residual amadoriase activity of a solution using thebuffer of the present invention, in the presence of a surfactant.

Examples of the buffer (buffer solution) which can be used in the kit(composition) of the present invention include a borate buffercontaining boric acid and/or a salt thereof; a Tris-hydrochloridebuffer; a phosphate buffer containing phosphoric acid and/or a saltthereof such as a potassium phosphate buffer or a sodium phosphatebuffer; an organic acid buffer containing an organic acid buffer and/ora salt thereof such as a tricarboxylate buffer containing tricarboxylicacid (buffer) and/or a salt thereof, a citrate buffer containing citricacid and/or a salt thereof; a monocarboxylate buffer containing amonocarboxylic acid (buffer) and/or a salt thereof such as an acetatebuffer containing an acetic acid (buffer) and/or a salt thereof.Examples of the buffer to be used in e.g., the kit of the presentinvention include Good's buffers including e.g., ACES(N-(2-acetamido)-2-aminoethanesulfonic acid), BES(N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), Bicin(N,N-bis(2-hydroxy-ethyl)glycine), Bis-Tris(bis(2-hydroxyethyl)iminotris (hydroxymethyl) methane), CHES(N-cyclohexyl-2-aminoethane sulfonic acid), EPPS(4-(2-hydroxyethyl)-1-piperazine-propanesulfonic acid), HEPES(4-2-hydroxyethyl-1-piperazine-ethanesulfonic acid), HEPPSO(N-(hydroxyethyl)piperazine-N′-2-hydroxy-propanesulfonic acid), MES(2-(N-morpholino)ethanesulfonic acid), MOPS(3-(N-morpholino)propanesulfonic acid), MOPSO(2-hydroxy-3-morpholino-propanesulfonic acid), PIPES(piperazine-N,N′-bis(2-ethanesulfonic acid)), POPSO(piperazine-1,4-bis(2-hydroxypropanesulfonic acid)), TAPS(N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid), TAPSO(3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid),TES (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid), Tricine(N-tris(hydroxymethyl)methylglycine) and/or salts thereof. Furthermore,a buffer containing a compound represented by the following formula(IV):

[where n may be 0, 1, 2 or 3; each R¹⁰ independently represents H, OH,—CH₂OH or —COOH], and/or a salt thereof, may be mentioned. Moreover, abuffer based on a dicarboxylic acid may be mentioned, including aphthalate buffer containing phthalic acid and/or a salt thereof; amaleate buffer containing maleic acid and/or a salt thereof; a fumaratebuffer containing fumaric acid and/or a salt thereof; a glutarate buffercontaining glutaric acid and/or a salt thereof; a citraconate buffercontaining citraconic acid and/or a salt thereof; a mesaconate buffercontaining mesaconic acid and/or a salt thereof; a malonate buffercontaining malonic acid and/or a salt thereof; a tartrate buffercontaining tartaric acid and/or a salt thereof; a succinate buffercontaining succinic acid and/or a salt thereof; an adipate buffercontaining adipic acid and/or a salt thereof; and a malate buffercontaining malic acid and/or a salt thereof. These, excluding HEPES andCHES, may serve as a buffer having the amadoriase stabilizing effect ofthe present invention. Examples of a preferable buffer having theamadoriase stabilizing effect of the present invention include, but notlimited to, a phosphate buffer, an ACES buffer, a citrate buffer, amalate buffer, an acetate buffer, a maleate buffer, a citraconatebuffer, a malonate buffer, a glutarate buffer, a tartrate buffer, and abuffer represented by formula (IV) such as MES buffer, MOPS buffer, andMOPSO buffer. These may be used alone or in combination of two or more.The buffer having the amadoriase stabilizing effect of the presentinvention may be used in combination with a substance (e.g., a buffernot having an amadoriase stabilizing effect) other than the abovebuffers. Examples of the salt include, but are not limited to, a sodiumsalt, a potassium salt, a magnesium salt, a calcium salt and an ammoniumsalt of a base compound.

The buffer of the present invention can be used in an appropriateconcentration in the kit or composition of the present invention. Ingeneral, the amount of buffer of the present invention to be added inthe kit or composition of the present invention can be calculated basedon the final concentration in a measurement solution. In one embodiment,the final concentration of the buffer of the present invention in ameasurement solution is the concentration at which a pH change that canoccur in the measurement solution is sufficiently buffered. In anotherembodiment, the final concentration of the buffer of the presentinvention in a measurement solution is the concentration at which theresidual activity of an amadoriase in a solution containing a surfactantbecomes 20% or higher, preferably 40% or higher, preferably 60% orhigher, and preferably 80% or higher. The final concentration of thebuffer of the present invention can be, for example, 1 mM or higher, 5mM or higher, 10 mM or higher, 20 mM or higher, for example 50 mM orhigher, 1M or lower, 500 mM or lower, 400 mM or lower, 300 mM or lower,200 mM or lower, 100 mM or lower, for example 1 mM to 1M, 5 mM to 500mM, 10 mM to 300 mM, for example 50 mM to 100 mM. When a phosphatebuffer is used as the buffer having the amadoriase stabilizing effect ofthe present invention, the concentration thereof can be 50 mM to 500 mM,for example, 50 mM to 300 mM and preferably 100 mM to 300 mM. When acitrate buffer, a malate buffer, maleic acid, a citraconate buffer, amalonate buffer, a glutarate buffer or a tartrate buffer is used as thebuffer of the present invention, the concentration thereof can be 5 mMto 500 mM, preferably 10 mM to 200 mM, for example, 10 mM to 100 mM.When a buffer represented by formula (IV) such as MES buffer, MOPSbuffer or MOPSO buffer is used as the buffer of the present invention,the concentration thereof can be 10 mM to 500 mM, for example, 100 mM to500 mM, for example 150 mM to 300 mM. When an ACES buffer is used as thebuffer of the present invention, the concentration thereof can be 200 mMto 1M, for example, 200 mM to 500 mM. As the buffer of the presentinvention, a plurality of buffers may be used in combination. The amountof buffer of the present invention to be used in a composition, if astabilizer is also added to the composition, may vary depending upon theamount of stabilizer.

(Stabilizer)

To the kit or composition of the present invention, a stabilizer, whichmaintains the residual activity of an amadoriase or lowers a reductionof the residual activity in the presence of a surfactant, can beappropriately added. In the present specification, the stabilizer refersto a substance, which maintains the residual activity of an amadoriaseor lowers a reduction of the residual activity in the presence of asurfactant. In the present specification, the expression stabilizerencompasses one or more stabilizers, unless otherwise indicated.Examples of the stabilizer to be contained in the kit or composition ofthe present invention include phosphoric acid, tricarboxylic acid (forexample, citric acid), dicarboxylic acid (for example, malic acid,maleic acid, citraconic acid, malonic acid, glutaric acid, tartaricacid), monocarboxylic acid (for example, acetic acid), a compoundrepresented by formula (IV) (for example, MES, MOPS, MOPSO), ammoniumsulfate, salts of these and any combination thereof.

The stabilizer of the present invention can be used in an appropriateconcentration in the kit or composition of the present invention. Ingeneral, the amount of stabilizer to be added to the kit or compositionof the present invention is calculated based on the final concentrationin the measurement solution. In one embodiment, the amount of stabilizeradded is the amount at which the residual activity of an amadoriase in asolution containing a surfactant is 35% or higher, 37.5% or higher,preferably 40% or higher, 45% or higher, 50% or higher, 55% or higher,preferably 60% or higher, 65% or higher, 70% or higher, 75% or higher,preferably 80% or higher, 85% or higher, 90% or higher or 95% or higher.The stabilizer of the present invention can be added to the kit orcomposition such that the final concentration in the measurementsolution becomes, for example, 0.1 mM to 100 mM, 0.2 mM to 100 mM, 0.5mM to 50 mM, 1 mM to 30 mM, 2 mM to 30 mM, 5 mM to 20 mM or 10 mM to 20mM. If a buffer is also added to the composition of the presentinvention, the amount of a stabilizer may vary depending on the amountof buffer. For example, to prevent pH change when a stabilizer is added,the type and amount of a buffer to be added may be appropriatelyselected and adjusted, or the pH of the stabilizer solution mayappropriately be adjusted.

Among the buffers of the present invention, in particular, a phosphatebuffer, a citrate buffer and MES buffer, when they are used in aconcentration, at which the pH of the solution can be kept at a constantlevel, more specifically, e.g., 100 mM for a phosphate buffer, e.g., 50mM for a citrate buffer, and e.g., 150 mM for MES buffer, an amadoriasestabilizing effect was observed. However, if the concentration ofphosphoric acid and/or a potassium salt thereof, citric acid and/or asodium salt thereof or MES and/or a sodium salt thereof to be added tothe composition are further reduced while maintaining the pH of thesolution within a constant range by use of HEPES having no amadoriasestabilizing effect as a pH buffer, the stabilization action of theresidual activity of an amadoriase in the presence of a surfactant wasobserved. The stabilization action was observed even at a lowerconcentration than those at which phosphoric acid and/or a potassiumsalt thereof, citric acid and/or a sodium salt thereof and MES and/or asodium salt thereof effectively exert a buffer action, morespecifically, 5 mM for phosphoric acid, 0.5 mM for citric acid and 20 mMfor MES. From this, it was confirmed that phosphoric acid and/or apotassium salt thereof, citric acid and/or a sodium salt thereof and MESand/or a sodium salt thereof have a stabilizing effect to maintainamadoriase activity apart from an amadoriase stabilizing effect as abuffer of the present invention. Such action may be referred to hereinas the amadoriase stabilizing effect of the stabilizer of the presentinvention, for convenience, in order to distinguish this from theamadoriase stabilizing effect of the buffer of the present invention.Thus, phosphoric acid and/or a potassium salt thereof, citric acidand/or a sodium salt thereof and MES and/or a sodium salt thereof havethe amadoriase stabilizing effect of the buffer of the present inventionas well as the amadoriase stabilizing effect of the stabilizer of thepresent invention. In other words, phosphoric acid and/or a potassiumsalt thereof, citric acid and/or a sodium salt thereof and MES and/or asodium salt thereof fall under not only the buffer of the presentinvention but also the stabilizer of the present invention.

Further, it was observed that maleic acid, citraconic acid, malonicacid, glutaric acid, tartaric acid, MOPS and MOPSO having amadoriasestabilizing effect exert an amadoriase stabilizing effect atconcentrations lower than the concentration at which a buffer action iseffectively exerted, such as 10 mM or 20 mM. These are compounds thatcan exert a buffer action at higher concentrations (e.g., 50 mM, 100 mM,150 mM). Thus, maleic acid, citraconic acid, malonic acid, glutaricacid, tartaric acid, MOPS, MOPSO and the compound represented by formula(IV) also fall under not only the buffer of the present invention butalso the stabilizer of the present invention.

When the kit or composition of the present invention comprises anamadoriase, a surfactant, a stabilizer and/or a buffer, these may beadded in any order to the kit or composition. Preferably, a stabilizerand/or buffer (if they are contained) are added and then a surfactant isadded to alleviate reduction of the residual activity of the amadoriase.

(Improvement of Surfactant Resistance of an Amadoriase of the PresentInvention)

The amadoriase of the present invention obtained by the aforementionedmeans has a mutation in its amino acid sequence by e.g., geneticmodification, with the result that the amadoriase has improvedsurfactant resistance, compared with an amadoriase prior to themodification. More specifically, the residual activity (%) of themodified amadoriase is improved 5 minutes after a particular surfactanttreatment, for example, after 0.01% (w/v) hexadecyltrimethylammoniumchloride (hereinafter referred to as, “CTAC”) is added at 30° C., in thereaction conditions described in the activity measurement method andsurfactant resistance evaluation method in the present specification,compared with the activity of an amadoriase prior to modification. Theresidual activity (%) herein refers to the ratio (%) of activity aftersurfactant treatment relative to the activity before the surfactanttreatment (regarded as 100). When the concentration of a surfactant inthe present specification is expressed by percentage, the percentagemeans % (w/v), unless indicated otherwise.

The degree of improvement of the residual activity (%) of a modifiedamadoriase of the present invention is not limited; however, forexample, the present invention encompasses a modified amadoriase havinga residual activity (%) of preferably 13% or higher, more preferably 15%or higher, most preferably 19% or higher, for example, 20% or higher,30% or higher, 40% or higher, 50% or higher, 60% or higher, 70% orhigher, 80% or higher, 90% or higher, 95% or higher or 99% or higherwhen it is measured before and after a mutation of the present inventionis introduced and subjected to a surfactant treatment. When amadoriasesbefore and after a mutation of the present invention is introduced aresubjected to a surfactant treatment and then the numerical values ofresidual activity (%) are compared, a modified amadoriase having aresidual activity improved by 2% or higher, preferably 9% or higher,most preferably 19% or higher, for example 20% or higher, 30% or higher,40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% orhigher, 90% or higher, 95% or higher or 99% or higher, is encompassed bythe present invention.

According to one embodiment, when a surfactant treatment is applied toan amadoriase before a mutation of the present invention is introduced,the amadoriase may completely lose its activity. In such a case, toevaluate improvement of the residual activity of an amadoriase (%) ofthe present invention to which a mutation of the present invention isintroduced, an amadoriase which will not completely lose its activityeven by a surfactant treatment is used and the residual activity of anamadoriase serving as a reference after a surfactant treatment may becompared with the residual activity of an amadoriase having a mutationintroduced therein after the surfactant treatment.

In situations, it may be difficult to evaluate the absolute surfactantresistance of variants merely based on whether numerical values of theresidual activity (%) and residual activity ratio are large or small,since relative evaluation results may differ depending on not onlytemperature conditions during measurement but also the degree ofsurfactant resistance of an amadoriase before introduction of amutation. However, it is possible to absolutely evaluate the surfactantresistance of variants by following the conditions of Examples of thepresent invention. Further, in order to readily select the amadoriase ofthe present invention, by selecting surfactant treatment conditions inwhich the residual activity of an amadoriase (%) before introduction ofa mutation is calculated to be sufficiently low, in general, the degreeof improvement of the residual activity (%) and the residual activityratio tend to be calculated to be high.

For example, when the amadoriase of the present invention produced byEscherichia coli JM109 (pKK223-3-CFP-T7/253K) strain encompassed by thepresent invention is mixed with 0.01% CTAC and subjected to a treatmentat 30° C. for 5 minutes, then the residual activity of amadoriase,CFP-T7, before introduction of the mutation of the present invention is69.9%; whereas, the residual activity thereof after introduction of themutation of the present invention is higher than 72%. When theamadoriase of the present invention produced by Escherichia coli JM109JM109 (pKK223-3-CFP-D7) strain is mixed with 0.04% CTAC and subjected toa treatment at 30° C. for 5 minutes, then the residual activity ofamadoriase, CFP-D, before introduction of the mutation of the presentinvention is 12.7%; whereas, the residual activity thereof afterintroduction of the mutation of the present invention is higher than15%. Likewise, an amadoriase improved in surfactant resistance issignificantly improved in storage property in e.g., enzyme-containingproducts, also, improves protease degradation efficiency of HbAlc, andincreases measurement sensitivity. Because of this, the amadoriase isstable when a strong surfactant is used and thus very useful from theperspective of the industry.

(Method of Measuring Activity of Amadoriase)

The activity of an amadoriase can be measured by various methods. Anexample of the method of measuring the activity of an amadoriase as usedherein is described below.

(Method of Measuring Activity of Amadoriase)

Examples of major methods for measuring the enzyme activity of theamadoriase of the present invention include a method of measuring theamount of hydrogen peroxide generated by enzyme reactions and a methodof measuring the amount of oxygen consumed in enzyme reactions. Anexample of the method of measuring the amount of hydrogen peroxide isdescribed below.

Hereinafter, when the activity of an amadoriase is measured in thepresent invention, fructosyl valine is used as a substrate, unlessotherwise specified. The titer of enzyme is defined such that, whenusing fructosyl valine as a substrate in measurement, the amount ofenzyme which generates 1 μmol of hydrogen peroxide per minute is(defined as) 1 U. A glycated amino acid such as fructosyl valine and aglycated peptide such as fructosyl-valyl histidine can be synthesizedand purified based on the method of Sakagami et al. (see, JP PatentPublication (Kokai) No. 2001-95598).

A. Preparation of Reagent

(1) Reagent 1: POD-4-AA Solution

Peroxidase (4.0 kU, manufactured by Kikkoman Corporation) and 100 mg of4-aminoantipyrine (manufactured by Tokyo Kasei Kogyo Co., Ltd.) aredissolved in 0.1 M potassium phosphate buffer (pH7.0) and the volume ofthe solution is fixed at 1 L.

(2) Reagent 2: TOOS Solution

TOOS (500 mg, sodium N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine,manufactured by Dojindo Laboratories) was dissolved in ion-exchangedwater and the volume of the solution is fixed at 100 ml.

(3) Reagent 3: Substrate Solution (150 mM; Final Concentration: 5 mM)

Fructosyl valine (417 mg) is dissolved in ion-exchanged water and thevolume of the solution is fixed at 10 ml.

B. Measurement Method

Reagent 1 (2.7 ml), reagent 2 (100 μl) and reagent 3 (100 μl) were mixedand preheated at 37° C. for 5 minutes. Then, to the mixture, an enzymesolution (100 μl) was added and thoroughly mixed. Thereafter, theabsorbance of the mixture at 555 nm is measured by a spectrophotometer(U-3010, manufactured by Hitachi High-Technologies). Measurement isperformed at 555 nm from one minute to three minutes and an absorbancechange per minute is specified as the measurement value. A controlsolution is prepared in the same manner as above except thation-exchanged water (100 μl) is used in place of reagent 3 (100 μl). Thenumber of micromoles of hydrogen peroxide generated per minute at 37° C.is specified as the activity unit (U) of the enzyme solution andcalculated in accordance with the following equation:

Activity (U/ml)={(ΔAs−ΔA0)×3.0×df}÷(39.2×0.5×0.1)

ΔAs: change in absorbance of reaction solution per minute

ΔA₀: change in absorbance of control solution per minute

39.2: millimole absorbance index (mM⁻¹·cm⁻¹) of quinoneimine dyegenerated by reaction

0.5: number of moles of quinoneimine dye generated by 1 mol hydrogenperoxide

df: dilution factor

(Method for Measuring Surfactant Resistance)

An amadoriase crude enzyme solution or an amadoriase purified sample isdiluted with a 30 mM MES/21 mM Tris buffer solution (pH6.5) so as tohave a concentration of about 1.0 U/ml. To this, CTAC (for example,manufactured by Tokyo Kasei Kogyo Co., Ltd.) is added so as to obtain afinal concentration 0.01% (w/v) or 0.04%. The resultant mixture isheated at 30° C. for 5 minutes. After heating, the mixture is diluteddouble with a 10 mM phosphate buffer (pH7.0) containing 0.15% BSA toprepare a sample. The enzyme activities of the sample before and after asurfactant treatment are measured by the method described in Section Babove. The ratio of activity of the sample after the surfactanttreatment relative to the activity of the sample before the surfactanttreatment (regarded as 100), i.e., the residual activity (%), isobtained. In this manner, surfactant resistance is evaluated.

(Method for Evaluating Buffer)

In the above surfactant resistance measuring method, the residualactivity of an amadoriase is measured by using various buffers in placeof a 30 mM MES/21 mM Tris buffer solution. In this manner, thecontribution of the buffer to the amadoriase residual activity can beevaluated. For example, in place of the 30 mM MES/21 mM Tris buffersolution (pH 6.5), e.g., a phosphate buffer solution (pH 7.0), a citratebuffer solution (pH 6.0), a HEPES buffer solution (pH 7.0) or an ACESbuffer solution (pH 7.0) can be used. Other conditions and procedure canbe the same as in the above surfactant resistance measuring method.

(Method for Evaluating Stabilizer)

In the above surfactant resistance measuring method, various stabilizersare added and the residual activity of an amadoriase is measured inorder to evaluate the effect of the stabilizers. In order to evaluatethe amadoriase stabilizing effect independently of contribution of thecompound (due to the buffer action) to the amadoriase residual activitywhen the stabilizers to be evaluated are compounds also having a bufferaction, a buffer having no amadoriase stabilizing effect is used at aconcentration sufficient to provide a buffer capacity to a solution (forexample, HEPES (pH 7.0) is used at 500 mM); while at the same time,stabilizers can be used at low concentrations insufficient to providebuffer capacity to the solution. The concentration sufficient to providea buffer capacity to a solution refers to the concentration at which pHis maintained within a predetermined range (for example pH5 to 10, pH6to 8) without having pH change due to other reagents added to thesolution. A concentration insufficient to provide a buffer capacity to asolution refers to the concentration at which pH changes by addition ofother reagents to the solution and pH falls outside a particular range.These concentrations vary depending upon the type and amount of otherreagents to be added to a solution; however, a person skilled in the artcan appropriately determine the concentration by conventional methods.Other conditions and procedure can be the same as in the abovesurfactant resistance measuring method.

(Action by Combined Use)

In order to evaluate the amadoriase stabilization action of a combineduse of the buffer of the present invention and the stabilizer of thepresent invention, the stabilizer and buffer can appropriately be addedwhile adjusting the concentrations thereof to a solution containing theamadoriase of the present invention and a surfactant, and then theresidual activity of the amadoriase can be measured. Other conditionsand procedure can be the same as in the above surfactant resistancemeasuring method.

The present invention will be more specifically described below withreference to Examples. However, these Examples are not intended in anyway to limit the technical scope of the present invention.

Example 1 (Mutation(s) for Improved Surfactant Resistance)

(1) Preparation of Recombinant Plasmid pKK223-3-CFP-T7 DNA

Escherichia coli strain JM109 (pKK223-3-CFP-T7) having a recombinantplasmid containing CFP-T7 gene (SEQ ID NO: 2) (see InternationalPublication No. WO 2007/125779) was inoculated in 2.5 ml of LB-ampmedium [1% (w/v) bactotrypton, 0.5% (w/v) peptone, 0.5% (w/v) NaCl, and50 μg/ml ampicillin] and subjected to shake culture at 37° C. for 20hours and a culture product was obtained.

The culture product was centrifuged at 7,000 rpm for 5 minutes tocollect strains. Then the recombinant plasmid pKK223-3-CFP-T7 wasextracted and purified therefrom using the QIAGEN tip-100 kit (QIAGEN),and 2.5 μg DNA of the recombinant plasmid pKK223-3-CFP-T7 was obtained.

(2) Site-Directed Modification Operation of DNA of Recombinant PlasmidpKK223-3-CFP-T7

PCR was carried out under conditions described below using obtained DNAof the recombinant plasmid pKK223-3-CFP-T7 as the template, syntheticoligonucleotides of SEQ ID NOs: 5 and 6, and KOD-Plus- (Toyobo Co.,Ltd.).

That is, 5 μl of 10×KOD-Plus-buffer, 5 μl of a dNTPs mixture in whicheach dNTP was adjusted at 2 mM, 2 μl of a 25 mM MgSO₄ solution, 50 ng ofDNA of pKK223-3-CFP-T7 as a template, 15 pmol each of the syntheticoligonucleotides, and 1 unit of KOD-Plus were mixed, and sterilizedwater was added thereto in order to bring the total amount of thesolution to 50 μl. The prepared reaction solution was subjected toincubation using a thermal cycler (manufactured by Eppendorf Co.) at 94°C. for 2 minutes, and a cycle of 94° C. for 15 seconds, 50° C. for 30seconds, and 68° C. for 6 minutes was then repeated 30 times.

A portion of the reaction solution was electrophoresed on 1.0% agarosegel, and the specific amplification of about 6,000 bp DNA was confirmed.The DNA thus obtained was treated with a restriction enzyme, DpnI (fromNew England Biolabs Co., Ltd.); after cleaving the remaining templateDNA, Escherichia coli JN109 was transformed therewith; and the resultanttransformants were spread on LB-amp agar medium. The grown colonies wereinoculated into LB-amp medium and subjected to shake culture, andplasmid DNA was isolated in the same manner as in (1) above. Thenucleotide sequence of DNA encoding an amadoriase in the plasmid wasdetermined using a multi-capillary DNA analysis system, AppliedBiosystems 3130×I Genetic Analyzer (from Life Technologies Co., Ltd.);as a result, a recombinant plasmid encoding a modified amadoriase inwhich asparagine at position 262 in the amino acid sequence described inSEQ ID NO: 1 was substituted with histidine (pKK223-3-CFP-T7-262H) wasobtained.

Subsequently, to substitute valine at position 257 in the amino acidsequence described in SEQ ID NO: 1 with cysteine, PCR reaction wascarried out under the same conditions as those described above using arecombinant plasmid, pKK223-3-CFP-T7 DNA, as a template, the syntheticoligonucleotides of SEQ ID NOS: 7 and 8, and KOD-Plus- (from Toyobo Co.,Ltd.) and the transformation of Escherichia coli JM109 and thedetermination of the nucleotide sequence of DNA encoding an amadoriasein the plasmid DNA carried on grown colonies were performed. As aresult, a recombinant plasmid encoding a modified amadoriase in whichvaline at position 257 in the amino acid sequence described in SEQ IDNO: 1 was substituted with cysteine (pKK223-3-CFP-T7-257C) was obtained.

Subsequently, to substitute valine at position 257 in the amino acidsequence described in SEQ ID NO: 1 with serine, PCR reaction was carriedout under the same conditions as those described above using arecombinant plasmid pKK223-3-CFP-T7 DNA as a template, the syntheticoligonucleotides of SEQ ID NOS: 8 and 9, and KOD-Plus- (from Toyobo Co.,Ltd.) and the transformation of Escherichia coli JM109 and thedetermination of the nucleotide sequence of DNA encoding an amadoriasein the plasmid DNA carried on grown colonies were performed. As aresult, a recombinant plasmid encoding a modified amadoriase in whichvaline at position 257 in the amino acid sequence described in SEQ IDNO: 1 was substituted with serine (pKK223-3-CFP-T7-257S) was obtained.

Subsequently, to substitute valine at position 257 in the amino acidsequence described in SEQ ID NO: 1 with threonine, PCR reaction wascarried out under the same conditions as those described above using arecombinant plasmid pKK223-3-CFP-T7 DNA as a template, syntheticoligonucleotides of SEQ ID NOS: 8 and 10, and KOD-Plus- (from ToyoboCo., Ltd.) and the transformation of Escherichia coli JM109 and thedetermination of the nucleotide sequence of DNA encoding an amadoriasein the plasmid DNA carried on grown colonies were performed. As aresult, a recombinant plasmid encoding a modified amadoriase in whichvaline at position 257 in the amino acid sequence described in SEQ IDNO: 1 was substituted with threonine (pKK223-3-CFP-T7-257T) wasobtained.

Subsequently, to substitute glutamic acid at position 253 in the aminoacid sequence described in SEQ ID NO: 1 with lysine, PCR reaction wascarried out under the same conditions as those described above using arecombinant plasmid pKK223-3-CFP-T7 DNA as a template, the syntheticoligonucleotides of SEQ ID NOS: 11 and 12, and KOD-Plus- (from ToyoboCo., Ltd.) and the transformation of Escherichia coli JM109 and thedetermination of the nucleotide sequence of DNA encoding an amadoriasein the plasmid DNA carried on grown colonies were performed. As aresult, a recombinant plasmid encoding a modified amadoriase in whichglutamic acid at position 253 in the amino acid sequence described inSEQ ID NO: 1 was substituted with lysine (pKK223-3-CFP-T7-253K) wasobtained.

Subsequently, to substitute glutamic acid at position 253 in the aminoacid sequence described in SEQ ID NO: 1 with arginine, PCR reaction wascarried out under the same conditions as those described above using arecombinant plasmid pKK223-3-CFP-T7 DNA as a template, the syntheticoligonucleotides of SEQ ID NOS: 12 and 13, and KOD-Plus- (from ToyoboCo., Ltd.) and the transformation of Escherichia coli JM109 and thedetermination of the nucleotide sequence of DNA encoding an amadoriasein the plasmid DNA carried on grown colonies were performed. As aresult, a recombinant plasmid encoding a modified amadoriase in whichglutamic acid at position 253 in the amino acid sequence described inSEQ ID NO: 1 was substituted with arginine (pKK223-3-CFP-T7-253R) wasobtained.

Subsequently, to substitute glutamine at position 337 in the amino acidsequence described in SEQ ID NO: 1 with lysine, PCR reaction was carriedout under the same conditions as those described above using arecombinant plasmid pKK223-3-CFP-T7-H1 DNA as a template, theoligonucleotides of SEQ ID NOS: 14 and 15, and KOD-Plus- (from ToyoboCo., Ltd.) and the transformation of Escherichia coli JM109 and thedetermination of the nucleotide sequence of DNA encoding an amadoriasein the plasmid DNA carried on grown colonies were performed. As aresult, a recombinant plasmid encoding a modified amadoriase in whichglutamine at position 337 in the amino acid sequence described in SEQ IDNO: 1 was substituted with lysine (pKK223-3-CFP-T7-337K) was obtained.

Subsequently, to substitute glutamic acid at position 340 in the aminoacid sequence described in SEQ ID NO: 1 with proline, PCR reaction wascarried out under the same conditions as those described above using arecombinant plasmid pKK223-3-CFP-T7 DNA as a template, theoligonucleotides of SEQ ID NOS: 16 and 17, and KOD-Plus- (from ToyoboCo., Ltd.) and the transformation of Escherichia coli JM109 and thedetermination of the nucleotide sequence of DNA encoding an amadoriasein the plasmid DNA carried on grown colonies were performed. As aresult, a recombinant plasmid encoding a modified amadoriase in whichglutamic acid at position 340 in the amino acid sequence described inSEQ ID NO: 1 was substituted with proline (pKK223-3-CFP-T7-340P) wasobtained.

Subsequently, to substitute glutamic acid at position 133 in the aminoacid sequence described in SEQ ID NO: 1 with alanine, PCR reaction wascarried out under the same conditions as those described above using arecombinant plasmid pKK223-3-CFP-T7 DNA as a template, the syntheticoligonucleotides of SEQ ID NOS: 18 and 19, and KOD-Plus- (from ToyoboCo., Ltd.) and the transformation of Escherichia coli JM109 and thedetermination of the nucleotide sequence of DNA encoding an amadoriasein the plasmid DNA carried on grown colonies were performed. As aresult, a recombinant plasmid encoding a modified amadoriase in whichglutamic acid at position 133 in the amino acid sequence described inSEQ ID NO: 1 was substituted with alanine (pKK223-3-CFP-T7-133A) wasobtained.

Subsequently, to substitute glutamic acid at position 133 in the aminoacid sequence described in SEQ ID NO: 1 with methionine, PCR reactionwas carried out under the same conditions as those described above usinga recombinant plasmid pKK223-3-CFP-T7 DNA as a template, the syntheticoligonucleotides of SEQ ID NOS: 19 and 20, and KOD-Plus- (from ToyoboCo., Ltd.) and the transformation of Escherichia coli JM109 and thedetermination of the nucleotide sequence of DNA encoding an amadoriasein the plasmid DNA carried on grown colonies were performed. As aresult, a recombinant plasmid encoding a modified amadoriase in whichglutamic acid at position 133 in the amino acid sequence described inSEQ ID NO: 1 was substituted with methionine (pKK223-3-CFP-T7-133M) wasobtained.

Subsequently, to substitute glutamic acid at position 133 in the aminoacid sequence described in SEQ ID NO: 1 with lysine, PCR reaction wascarried out under the same conditions as those described above using arecombinant plasmid pKK223-3-CFP-T7-H1 DNA as a template, the syntheticoligonucleotides of SEQ ID NOS: 19 and 21, and KOD-Plus-(from ToyoboCo., Ltd.) and the transformation of Escherichia coli JM109 and thedetermination of the nucleotide sequence of DNA encoding an amadoriasein the plasmid DNA carried on grown colonies were performed. As aresult, a recombinant plasmid encoding a modified amadoriase in whichglutamic acid at position 133 in the amino acid sequence described inSEQ ID NO: 1 was substituted with lysine (pKK223-3-CFP-T7-133K) wasobtained.

Subsequently, to substitute glutamic acid at position 44 in the aminoacid sequence described in SEQ ID NO: 1 with proline, PCR reaction wascarried out under the same conditions as those described above using arecombinant plasmid pKK223-3-CFP-T7 DNA as a template, the syntheticoligonucleotides of SEQ ID NOS: 22 and 23, and KOD-Plus- (from ToyoboCo., Ltd.) and the transformation of Escherichia coli JM109 and thedetermination of the nucleotide sequence of DNA encoding an amadoriasein the plasmid DNA carried on grown colonies were performed. As aresult, a recombinant plasmid encoding a modified amadoriase in whichglutamic acid at position 44 in the amino acid sequence described in SEQID NO: 1 was substituted with proline (pKK223-3-CFP-T7-44P) wasobtained.

Subsequently, to substitute glycine at position 256 in the amino acidsequence described in SEQ ID NO: 1 with lysine, PCR reaction was carriedout under the same conditions as those described above using arecombinant plasmid pKK223-3-CFP-T7 DNA as a template, the syntheticoligonucleotides of SEQ ID NOS: 8 and 24, and KOD-Plus- (from ToyoboCo., Ltd.) and the transformation of Escherichia coli JM109 and thedetermination of the nucleotide sequence of DNA encoding an amadoriasein the plasmid DNA carried on grown colonies were performed. As aresult, a recombinant plasmid encoding a modified amadoriase in whichglycine at position 256 in the amino acid sequence described in SEQ IDNO: 1 was substituted with lysine (pKK223-3-CFP-T7-256K) was obtained.

Subsequently, to substitute glycine at position 256 in the amino acidsequence described in SEQ ID NO: 1 with arginine, PCR reaction wascarried out under the same conditions as those described above using arecombinant plasmid pKK223-3-CFP-T7 DNA as a template, the syntheticoligonucleotides of SEQ ID NOS: 8 and 25, and KOD-Plus- (from ToyoboCo., Ltd.) and the transformation of Escherichia coli JM109 and thedetermination of the nucleotide sequence of DNA encoding an amadoriasein the plasmid DNA carried on grown colonies were performed. As aresult, a recombinant plasmid encoding a modified amadoriase in whichglycine at position 256 in the amino acid sequence described in SEQ IDNO: 1 was substituted with arginine (pKK223-3-CFP-T7-256R) was obtained.

Subsequently, to substitute glutamic acid at position 81 in the aminoacid sequence described in SEQ ID NO: 1 with lysine, PCR reaction wascarried out under the same conditions as those described above using arecombinant plasmid pKK223-3-CFP-T7 DNA as a template, the syntheticoligonucleotides of SEQ ID NOS: 26 and 27, and KOD-Plus- (from ToyoboCo., Ltd.) and the transformation of Escherichia coli JM109 and thedetermination of the nucleotide sequence of DNA encoding an amadoriasein the plasmid DNA carried on grown colonies were performed. As aresult, a recombinant plasmid encoding a modified amadoriase in whichglutamic acid at position 81 in the amino acid sequence described in SEQID NO: 1 was substituted with lysine (pKK223-3-CFP-T7-81K) was obtained.

Subsequently, to substitute aspartic acid at position 129 in the aminoacid sequence described in SEQ ID NO: 1 with lysine, PCR reaction wascarried out under the same conditions as those described above using arecombinant plasmid pKK223-3-CFP-T7 DNA as a template, the syntheticoligonucleotides of SEQ ID NOS: 46 and 47, and KOD-Plus- (from ToyoboCo., Ltd.) and the transformation of Escherichia coli JM109 and thedetermination of the nucleotide sequence of DNA encoding an amadoriasein the plasmid DNA carried on grown colonies were performed. As aresult, a recombinant plasmid encoding a modified amadoriase in whichaspartic acid at position 129 in the amino acid sequence described inSEQ ID NO: 1 was substituted with lysine (pKK223-3-CFP-T7-129K) wasobtained.

Subsequently, to substitute aspartic acid at position 132 in the aminoacid sequence described in SEQ ID NO: 1 with lysine, PCR reaction wascarried out under the same conditions as those described above using arecombinant plasmid pKK223-3-CFP-T7 DNA as a template, the syntheticoligonucleotides of SEQ ID NOS: 19 and 48, and KOD-Plus- (from ToyoboCo., Ltd.) and the transformation of Escherichia coli JM109 and thedetermination of the nucleotide sequence of DNA encoding an amadoriasein the plasmid DNA carried on grown colonies were performed. As aresult, a recombinant plasmid encoding a modified amadoriase in whichaspartic acid at position 132 in the amino acid sequence described inSEQ ID NO: 1 was substituted with lysine (pKK223-3-CFP-T7-132K) wasobtained.

Subsequently, to substitute glutamic acid at position 231 in the aminoacid sequence described in SEQ ID NO: 1 with lysine, PCR reaction wascarried out under the same conditions as those described above using arecombinant plasmid pKK223-3-CFP-T7 DNA as a template, the syntheticoligonucleotides of SEQ ID NOS: 49 and 50, and KOD-Plus- (from ToyoboCo., Ltd.) and the transformation of Escherichia coli JM109 and thedetermination of the nucleotide sequence of DNA encoding the asparaginein the plasmid DNA carried on grown colonies were performed. As aresult, a recombinant plasmid encoding a modified amadoriase in whichglutamic acid at position 231 in the amino acid sequence described inSEQ ID NO: 1 was substituted with lysine (pKK223-3-CFP-T7-231K) wasobtained.

Subsequently, to substitute aspartic acid at position 232 in the aminoacid sequence described in SEQ ID NO: 1 with lysine, PCR reaction wascarried out under the same conditions as those described above using arecombinant plasmid pKK223-3-CFP-T7 DNA as a template, the syntheticoligonucleotides of SEQ ID NOS: 50 and 51, and KOD-Plus- (from ToyoboCo., Ltd.) and the transformation of Escherichia coli JM109 and thedetermination of the nucleotide sequence of DNA encoding an amadoriasein the plasmid DNA carried on grown colonies were performed. As aresult, a recombinant plasmid encoding a modified amadoriase in whichaspartic acid at position 232 in the amino acid sequence described inSEQ ID NO: 1 was substituted with lysine (pKK223-3-CFP-T7-232K) wasobtained.

Subsequently, to substitute glutamic acid at position 249 in the aminoacid sequence described in SEQ ID NO: 1 with lysine, PCR reaction wascarried out under the same conditions as those described above using arecombinant plasmid pKK223-3-CFP-T7 DNA as a template, the syntheticoligonucleotides of SEQ ID NOS: 52 and 53, and KOD-Plus- (from ToyoboCo., Ltd.) and the transformation of Escherichia coli JM109 and thedetermination of the nucleotide sequence of DNA encoding the amadoriasein the plasmid DNA carried on grown colonies were performed. As aresult, a recombinant plasmid encoding a modified amadoriase in whichglutamic acid at position 249 in the amino acid sequence described inSEQ ID NO: 1 was substituted with lysine (pKK223-3-CFP-T7-249K) wasobtained.

(3) Production of Various Modified Amadoriase

Escherichia coli strain JM109 holding each of the above recombinantplasmids obtained by the above procedures was cultured at 30° C. for 16hours in 3 ml of LB-amp medium containing 0.1 mM IPTG. Then, thebacterial bodies of each strain were washed with a 0.01 M phosphatebuffer solution (pH 7.0), ultrasonically disintegrated, and centrifugedat 15,000 rpm for 10 minutes, and 1.5 ml of each crude enzyme solutionwas prepared.

(4) Evaluation of Surfactant Resistance of Various Modified Amadoriase

Using each crude enzyme solution thus prepared as a sample, the finalconcentration of CTAC was set at 0.01% to evaluate the surfactantresistance of each of the modified amadoriases according to the abovemeasurement method for surfactant resistance. The results are shown inTable 1-1. In this respect, it was confirmed that when the warmed samplewas again measured for activity 30 minutes after 2-fold dilution in aBSA solution, there was no change in the activity value. In Table 1-1,CFP-T7 indicates an amadoriase derived from Escherichia coli strainJM109 (pKK223-3-CFP-T7). Since CFP-T7 as an amadoriase derived fromEscherichia coli strain JM109 (pKK223-3-CFP-T7) was used as the enzymeto which mutations were introduced, the mutation points alreadyintroduced into CFP-T7 are not included in the description of the “AminoAcid Mutation” described in the table.

TABLE 1-1 Residual Amino Acid Activity Plasmid Enzyme Mutation (%)pKK223-3-CFP-T7 CFP-T7 None 69.9 pKK223-3-CFP-T7/262H CFP-T7/262H N262H94.6 pKK223-3-CFP-T7/257C CFP-T7/257C V257C 72.8 pKK223-3-CFP-T7/257SCFP-T7/257S V257S 84.9 pKK223-3-CFP-T7/257T CFP-T7/257T V257T 86.4pKK223-3-CFP-T7/253K CFP-T7/253K E253K 99.2 pKK223-3-CFP-T7/253RCFP-T7/253R E253R 94.5 pKK223-3-CFP-T7/337K CFP-T7/337K Q337K 89.7pKK223-3-CFP-T7/340P CFP-T7/340P E340P 93.6 pKK223-3-CFP-T7/133ACFP-T7/133A E133A 79.0 pKK223-3-CFP-T7/133M CFP-T7/133M E133M 72.7pKK223-3-CFP-T7/133K CFP-T7/133K E133K 97.5 pKK223-3-CFP-T7/44PCFP-T7/44P E44P 76.0 pKK223-3-CFP-T7/256K CFP-T7/256K G256K 85.2pKK223-3-CFP-T7/256R CFP-T7/256R G256R 96.6 pKK223-3-CFP-T7/81KCFP-T7/81K E81K 72.2 pKK223-3-CFP-T7/129K CFP-T7/129K D129K 97.1pKK223-3-CFP-T7/132K CFP-T7/132K D132K 97.1 pKK223-3-CFP-T7/231KCFP-T7/231K E231K 96.6 pKK223-3-CFP-T7/232K CFP-T7/232K D232K 96.6pKK223-3-CFP-T7/249K CFP-T7/249K E249K 100

As shown in Table 1-1, the residual activity of CFP-T7 was 69.9% underthe conditions of this Example. In contrast, the residual activity wasenhanced to 72% or more (79% or more in notable instances and 89% ormore in more notable instances) in the 15 variants obtained by theintroduction of site-specific mutation, i.e., amadoriases in each ofwhich asparagine at position 262 in CFP-T7 is mutated to histidine,valine at position 257 to cysteine, serine, or threonine, glutamic acidat position 253 to lysine or arginine, glutamine at position 337 tolysine, glutamic acid at position 340 to proline, glutamic acid atposition 44 to proline, glutamic acid at position 133 to alanine,methionine, or lysine, glycine at position 256 to lysine or arginine,glutamic acid at position 81 to lysine, aspartic acid at position 129 tolysine, aspartic acid at position 132 to lysine, glutamic acid atposition 231 to lysine, aspartic acid at position 232 to lysine, orglutamic acid at position 249 to lysine. Thus, these mutation points areeach confirmed to be a mutation point for enhancing the surfactantresistance of an amadoriase.

The substitution of the amino acid at each of positions 253 and 256 inCFP-T7 with each of basic amino acid residues lysine and arginine wasobserved to enhance surfactant resistance. Thus, it is believed that thesubstitution of the amino acid at each of positions 81, 129, 132, 133,231, 232, 249, and 337 with arginine which is a basic amino acid residuewill enhance surfactant resistance, as is the case with lysine.

(5) CcFX Derived from Curvularia clavata

SEQ ID NO: 37 is the amino acid sequence of a ketoamine oxidase derivedfrom Curvularia clavata (hereinafter referred to as CcFX) (InternationalPublication No. WO 2004/104203). A gene (SEQ ID NO: 55) encoding theamino acid sequence of SEQ ID NO: 37 was obtained by totallysynthesizing cDNA by PCR of a gene fragment as a conventional method(the stop codon TAA is contained). At this time, an EcoRI site and aHindIII site were added to the 5′-end and the 3′-end of SEQ ID NO: 55,respectively. The full-length amino acid sequence deduced based on thecloned gene sequence was confirmed to be consistent with the sequence ofCcFX in FIG. 1A-1C. Subsequently, to express the gene of SEQ ID NO: 55obtained in Escherichia coli, the following procedures were performed.The gene totally synthesized above was first treated with tworestriction enzymes for the EcoRI site and the HindIII site (from TakaraBio Inc.) and inserted into the EcoRI-HindIII site of the pKK-223-3vector (from GE Healthcare Co., Ltd.) to provide a recombinant plasmid,pKK223-3-CcFX. This plasmid was transformed into Escherichia coli strainJM109 under the same conditions as those described above to provideEscherichia coli strain JM109 (pKK223-3-CcFX).

Then, mutations for enhancing surfactant resistance were introduced intoCcFX. More specifically, mutation was introduced into positions 129,132, 133, 229, 230, 247, 251, 254, and 335 in CcFX as positionscorresponding to positions 129, 132, 133, 231, 232, 249, 253, 256, and337 in the amadoriase derived from the genus Coniochaeta (CFP-T7).

Using a recombinant plasmid containing CcFX gene (SEQ ID NO: 55) as astarting plasmid, various variants were produced as in the proceduresdescribed in (1) and (2) above in Escherichia coli strain JM109(pKK223-3-CcFX) having the plasmid. The sequences of primers used forthe mutation introduction are as shown in SEQ ID NOS: 56 to 74. Then,modified amadoriases were produced by the procedure described in (3)above. Subsequently, the surfactant resistance of the modifiedamadoriases was evaluated according to the measurement method forsurfactant resistance described in (4) although under surfactanttreatment conditions in which the amadoriases were each diluted in a 20mM potassium phosphate buffer solution (pH 7.0) and mixed with 0.01%CTAC. The results are shown in Table 1-2. In this respect, it wasconfirmed that when the warmed sample was again measured for activity 30minutes after 2-fold dilution in a BSA solution, there was no change inthe activity value.

TABLE 1-2 SEQ ID Amino NO of Acid Oligonu- Residual Muta- cleotideActivity Plasmid Enzyme tion Used (%) pKK223-3-CcFX CcFX None None 27.4pKK223-3-CcFX/129K CcFX/129K D129K 56.57 37.6 pKK223-3-CcFX/132KCcFX/132K D132K 58.61 34.8 pKK223-3-CcFX/133K CcFX/133K E133K 59.61 92.4pKK223-3-CcFX/133A CcFX/133A E133A 60.61 43.0 pKK223-3-CcFX/229KCcFX/229K E229K 62.64 56.4 pKK223-3-CcFX/230K CcFX/230K D230K 63.64 44.1pKK223-3-CcFX/247K CcFX/247K E247K 65.66 88.0 pKK223-3-CcFX/251KCcFX/251K E251K 67.68 64.3 pKK223-3-CcFX/251R CcFX/251R E251R 68.69 41.6pKK223-3-CcFX/254K CcFX/254K N254K 70.71 63.8 pKK223-3-CcFX/335KCcFX/335K T335K 72.73 58.3 pKK223-3-CcFX/335R CcFX/335R T335R 73.74 38.5

As shown in Table 1-2, the residual activity of CcFX was 27.4% under theconditions of this Example. In contrast, the residual activity wasenhanced to 34% or more (56% or more in notable instances and 64% ormore in more notable instances) in the 12 variant amadoriases obtainedby the introduction of site-specific mutation.

As above, when the mutation confirmed to enhance surfactant resistancefor CFP-T7 was introduced into the corresponding positions in CcFX,similar improvements of surfactant resistance were confirmed asdescribed above. Thus, the effect of the introduction of these mutationsis not limited to amadoriases derived from a specific species and theintroduction also has the effect of improving the surfactant resistanceof various amadoriases by introducing mutation at the correspondingpositions.

Incidentally, the amadoriase derived from the genus Coniochaeta hasabout 80% amino acid sequence identity to the ketoamine oxidase derivedfrom Curvularia clavata. Hence, amadoriases derived from other specieshaving 80% or more amino acid sequence identity to the amadoriasederived from the genus Coniochaeta or ketoamine oxidase derived fromCurvularia clavata are thought to have improved (enhanced) surfactantresistance by the introduction of mutation into positions correspondingto the above positions.

The substitution of the amino acid at each of positions 251 and 335 inCcFX with lysine or arginine was observed to enhance surfactantresistance. From these results, surfactant resistance is thought to beenhanced by the substitution of the amino acid at each of positions 81,129, 132, 133, 229, 230, 247, 251, 254, and 335 in CcFX with lysine orarginine which are basic amino acid residues. The same applies tovarious other amadoriases.

The substitution of the amino acid at position 133 in CFP-T7 withalanine or methionine or the substitution of the amino acid at position133 in CcFX with alanine was observed to enhance surfactant resistance.From these results, surfactant resistance is thought to be enhanced bythe substitution of the amino acid at each of position 133 in CFP-T7 andposition 133 in CcFX to alanine, methionine, valine, isoleucine,leucine, phenylalanine, tryptophan, or proline as a hydrophobic aminoacid residue. The same applies to various other amadoriases.

Without wishing to be being bound by any particular theory, themechanism by which the variant amadoriase of the present inventionbecomes resistant to a surfactant is thought, for example, to be asfollows. That is, the substitution of an acidic amino acid in anamadoriase with a hydrophobic amino acid or a basic amino acid isconsidered to reduce the affinity between the amadoriase and a cationicsurfactant and protect the amadoriase from the denaturing action of thesurfactant. In particular, the introduction of lysine or arginine, whichare basic amino acid residues, is considered to cause the basic aminoacid residue to repel a cationic surfactant to further protect anamadoriase from the denaturing action of a surfactant.

These mutation points of the present invention not only are effective insingle mutation, but also are expected to contribute to creatingvariants having practical advantages by combining with various knownmutations or combining the mutations of the present invention with eachother.

Example 2 (Accumulation of Mutation for Improved Surfactant Resistance)

Based on the findings of mutations for enhancing surfactant resistanceobtained in Example 1, multiple variants (a double variant, a triplevariant, a quadruple variant, a quintuple variant, a sextuple variant,or a septuple variant) were tested to combine and accumulate thesemutations in order to obtain an amadoriase having further increasedsurfactant resistance.

SEQ ID NO: 3 is the amino acid sequence of an amadoriase derived fromthe genus Coniochaeta into which a mutation for improving substratespecificity (E98A) and mutations for enhancing heat stability (F43Y,G184D, deletion of 3 carboxy-terminal amino acid residues) wereintroduced (hereinafter indicated with “CFP-D”), and is encoded by thegene of SEQ ID NO: 4. Mutations for enhancing surfactant resistance wereaccumulated using plasmid DNA in which CFP-D gene was inserted intopKK223-3 vector as a template. PCR reaction was carried out under thesame conditions as those in (2) above using the syntheticoligonucleotides of SEQ ID NOS: 5, 6, 7, 16, 17, 18, 19, 23, 28, 29, 30,31, 32, 33, 46, 47, 50, 51, 52, and 54, and KOD-Plus- (from Toyobo Co.,Ltd.) and the transformation of Escherichia coli strain JM109 and thedetermination of the nucleotide sequence of DNA encoding the amadoriasein the plasmid DNA carried on grown colonies were performed.

These procedures provided pKK223-3-CFP-D1 as a variant (mutant) in whichglutamic acid at position 44 was substituted with proline;pKK223-3-CFP-D2 as a double variant in which glutamic acid at position44 was substituted with proline and glutamic acid at position 340 wassubstituted with proline; pKK223-3-CFP-D3 as a triple variant in whichglutamic acid at position 44 was substituted with proline, asparagine atposition 262 was substituted with histidine, and glutamic acid atposition 340 was substituted with proline: pKK223-3-CFP-D4 as aquadruple variant in which glutamic acid at position 44 was substitutedwith proline, valine at position 257 was substituted with cysteine,asparagine at position 262 was substituted with histidine, and glutamicacid at position 340 was substituted with proline; pKK223-3-CFP-D4/232Kas a quintuple variant in which glutamic acid at position 44 wassubstituted with proline, valine at position 257 was substituted withcysteine, asparagine at position 262 was substituted with histidine,glutamic acid at position 340 was substituted with proline, andasparagine at position 232 was substituted with lysine;pKK223-3-CFP-D4/249K as a quintuple variant in which glutamic acid atposition 44 was substituted with proline, valine at position 257 wassubstituted with cysteine, asparagine at position 262 was substitutedwith histidine, glutamic acid at position 340 was substituted withproline, and glutamic acid at position 249 was substituted with lysine;pKK223-3-CFP-D5 as a quintuple variant in which glutamic acid atposition 44 was substituted with proline, glutamic acid at position 253was substituted with lysine, valine at position 257 was substituted withcysteine, asparagine at position 262 was substituted with histidine, andglutamic acid at position 340 was substituted with proline;pKK223-3-CFP-D5/129K as a sextuple variant in which glutamic acid atposition 44 was substituted with proline, glutamic acid at position 253was substituted with lysine, valine at position 257 was substituted withcysteine, asparagine at position 262 was substituted with histidine,glutamic acid at position 340 was substituted with proline, and asparticacid at position 129 was substituted with lysine; a pKK223-3-CFP-D6 as asextuple variant in which glutamic acid at position 44 was substitutedwith proline, glutamic acid at position 133 was substituted withalanine, glutamic acid at position 253 was substituted with lysine,valine at position 257 was substituted with cysteine, asparagine atposition 262 was substituted with histidine, and glutamic acid atposition 340 was substituted with proline; and pKK223-3-CFP-D7 as aseptuple variant in which glutamic acid at position 44 was substitutedwith proline, glutamic acid at position 133 was substituted withalanine, glutamic acid at position 253 was substituted with lysine,valine at position 257 was substituted with cysteine, asparagine atposition 262 was substituted with histidine, glutamine at position 337was substituted with lysine, and glutamic acid at position 340 wassubstituted with proline.

Then, Escherichia coli strain JM109 was transformed under the sameconditions as those described above and Escherichia coli strain JM109(pKK223-3-CFP-D), Escherichia coli strain JM109 (pKK223-3-CFP-D1),Escherichia coli strain JM109 (pKK223-3-CFP-D2), Escherichia coli strainJM109 (pKK223-3-CFP-D3), Escherichia coli strain JM109(pKK223-3-CFP-D4), Escherichia coli strain JM109 (pKK223-3-CFP-D4/232K),Escherichia coli strain JM109 (pKK223-3-CFP-D4/249K), Escherichia colistrain JM109 (pKK223-3-CFP-D5), Escherichia coli strain JM109(pKK223-3-CFP-D5/129K), Escherichia coli strain JM109 (pKK223-3-CFP-D6),and Escherichia coli strain JM109 (pKK223-3-CFP-D7) were obtained.

The Escherichia coli strains having the ability to produce modifiedamadoriases obtained as described above, i.e., Escherichia coli strainJM109 (pKK223-3-CFP-T7), Escherichia coli strain JM109 (pKK223-3-CFP-D),Escherichia coli strain JM109 (pKK223-3-CFP-D1), Escherichia coli strainJM109 (pKK223-3-CFP-D2), Escherichia coli strain JM109(pKK223-3-CFP-D3), Escherichia coli strain JM109 (pKK223-3-CFP-D4),Escherichia coli strain JM109 (pKK223-3-CFP-D4/232K), Escherichia colistrain JM109 (pKK223-3-CFP-D4/249K), Escherichia coli strain JM109(pKK223-3-CFP-D5), Escherichia coli strain JM109 (pKK223-3-CFP-D5/129K),Escherichia coli strain JM109 (pKK223-3-CFP-D6), and Escherichia colistrain JM109 (pKK223-3-CFP-D7), were cultured by the above method toprepare 1.5 ml of a crude enzyme solution of each of the modifiedamadoriases. Using the resultant crude enzyme solutions as samples, thesurfactant resistance of the modified amadoriases was evaluatedaccording to the measurement method for surfactant resistance describedin (4) above although under more stringent surfactant treatmentconditions in which treatment was altered to be mixing with 0.04% CTAC.The results are shown in Table 2-1. In this respect, it was confirmedthat when the warmed sample was again measured for activity 30 minutesafter 2-fold dilution in a BSA solution, there was no change in theactivity value.

TABLE 2-1 SEQ ID NO of Residual Plasmid as Amino Acid OligonucleotideActivity Plasmid Template Enzyme Mutation Used (%) pKK223-3- None CFP-T7None None 2.27 CFP-T7 pKK223-3- None CFP-D None None 12.7 CFP-DpKK223-3- pKK223-3- CFP-D1 E44P 23.28 15.2 CFP-D1 CFP-D pKK223-3-pKK223-3- CFP-D2 E44P/E340P 16.17 37.1 CFP-D2 CFP-D1 pKK223-3- pKK223-3-CFP-D3 E44P/N262H/E340P 5.6 51.4 CFP-D3 CFP-D2 pKK223-3- pKK223-3-CFP-D4 E44P/V257C/N262H/ 7.29 60.7 CFP-D4 CFP-D3 E340P pKK223-3-pKK223-3- CFP-D4/232K E44P/V257C/N262H/ 50.51 66.2 CFP-D4/232K CFP-D3E340P/D232K pKK223-3- pKK223-3- CFP-D4/249K E44P/V257C/N262H/ 52.54 91.0CFP-D4/249K CFP-D3 E340P/E249K pKK223-3- pKK223-3- CFP-D5E44P/E253K/V257C/ 30.31 95.6 CFP-D5 CFP-D4 N262H/E340P pKK223-3-pKK223-3- CFP-D5/129K E44P/E253K/V257C/ 46.47 98.1 CFP-D5/129K CFP-D4N262H/E340P/D129K pKK223-3- pKK223-3- CFP-D6 E44P/E133A/E253K/ 18.1999.2 CFP-D6 CFP-D5 V257C/N262H/E340P pKK223-3- pKK223-3- CFP-D7E44P/E133A/E253K/ 32.33 100 CFP-D7 CFP-D6 V257C/N262H/Q337K/ E340P

As shown in Table 2-1, the residual activity of CFP-T7 was merely 2.27%under the conditions of this Example. It was confirmed that conventionalamadoriases virtually lost almost all activity under such harshconditions.

In contrast, all of the multiple variants prepared by the variouscombinations of the single mutations identified in Example 1 hadsignificantly enhanced residual activities. In particular, the residualactivity of the double variant in which glutamic acid at position 44 wassubstituted with proline and glutamic acid at position 340 wassubstituted with proline was 37.1% and was enhanced compared to that ofCFP-T7. The residual activity of the triple variant in which asparagineat position 262 was substitutes by histidine in addition to the priormutation was 51.4% and was further enhanced compared to that of CFP-T7.The residual activity of the quadruple variant in which valine atposition 257 was substituted with cysteine in addition to the priormutation was 60.7% and significantly enhanced compared to that ofCFP-T7. The residual activity of the quintuple variant in which glutamicacid at position 253 was substituted with lysine in addition to theprior mutation was 95.6%; the residual activity of the sextuple variantin which glutamic acid at position 133 was substituted with alanine inaddition to the prior mutation was 99.2%; the residual activity of theseptuple variant in which glutamine at position 337 was substituted withlysine in addition to the prior mutation was 100% and was significantlyenhanced compared to CFP-T7, and there was almost no inactivation of theamadoriase due to CTAC. The residual activity of the quintuple variantin which aspartic acid at position 232 in the quadruple variant CFP-D4was further substituted with lysine was 66.2%; the residual activity ofthe quintuple variant in which glutamic acid at position 249 in thequadruple variant CFP-D4 was substituted with lysine was 91.0%; theresidual activity of the sextuple variant in which aspartic acid atposition 129 in the quintuple variant CFP-D5 was substituted with lysinewas 98.1% and was significantly enhanced compared to that of CFP-T7, andthere was almost no inactivation of the amadoriase due to CTAC.

In addition, each time mutations were accumulated into CFP-D, thesurfactant resistance of the resultant further multiple variant wasincrementally enhanced, demonstrating that the mutation points of thepresent invention identified in Example 1 could appropriately becombined to produce an amadoriase having further excellent surfactantresistance.

Next, mutations for enhancement of surfactant resistant were accumulatedusing a plasmid DNA in which CcFX gene was inserted into pKK223-3 vectoras a template. The procedures were carried out as described above exceptfor the point that the CcFX gene was used instead of the CFP-D gene. PCRreaction was carried out under the same conditions as those in (2) aboveusing synthetic oligonucleotides (SEQ ID NOS: 72 and 73) andKOD-Plus-(from Toyobo Co., Ltd.) and the transformation of Escherichiacoli strain JM109 and the determination of the nucleotide sequence ofDNA encoding the amadoriase in the plasmid DNA carried on grown colonieswere performed.

These procedures provided pKK223-3-CcFX/132K/335K as a variant in whichaspartic acid at position 132 was substituted with lysine and threonineat position 335 was substituted with lysine; pKK223-3-CcFX/133A/335K asa variant in which glutamic acid at position 133 was substituted withalanine and threonine at position 335 was substituted with lysine;pKK223-3-CcFX/229K/335K as a variant in which glutamic acid at position229 was substituted with lysine and threonine at position 335 wassubstituted with lysine; and pKK223-3-CcFX/251K/335K as a variant inwhich glutamic acid at position 251 was substituted with lysine andthreonine at position 335 was substituted with lysine. Then, Escherichiacoli strain JM109 was transformed under the same conditions as thosedescribed above; the resultant transformant strains were cultured by theabove method; and 1.5 ml each of the crude enzyme solutions of themodified amadoriases were prepared. Using the resultant crude enzymesolutions as samples, the surfactant resistance of the modifiedamadoriases was evaluated according to the measurement method forsurfactant resistance described in (4) although under surfactanttreatment conditions in which the amadoriases were each diluted in a 20mM potassium phosphate buffer solution (pH 7.0) and mixed with 0.01%CTAC. The results are shown in Table 2-2. In this respect, it wasconfirmed that when the warmed sample was again measured for activity 30minutes after 2-fold dilution in a BSA solution, there was no change inthe activity value.

TABLE 2-2 SEQ ID NO of Residual Plasmid as Amino Acid OligonucleotideActivity Plasmid Template Enzyme Mutation Used (%) pKK223-3- pKK223-3-CcFX/ D132K/T335K 72.73 82.8 CcFX/ CcFX/ 132K/335K 132K/335K 132KpKK223-3- pKK223-3- CcFX/ E133A/T335K 86.0 CcFX/ CcFX/ 133A/335K133A/335K 133A pKK223-3- pKK223-3- CcFX/ E229K/T335K 65.3 CcFX/ CcFX/229K/335K 229K/335K 229K pKK223-3- pKK223-3- CcFX/ E251K/T335K 90.5CcFX/ CcFX/ 251K/335K 251K/335K 251K

As shown in Tables 1-2 and 2-2, the residual activity of CcFX was 27.4%under the conditions of this Example, whereas all of the double variantsprepared by combining the single mutations identified in Example 1 hadsignificantly enhanced residual activities. The surfactant resistance ofthe double variants of CcFX also was enhanced compared to that of thesingle variants of CcFX in Table 1-2, also confirming that regardless ofthe type of an amadoriase enzyme, the effect of mutation wasaccumulated.

Example 3-1 (Evaluation for Surfactant TTAC)

Tetradecyltrimethylammonium chloride (hereinafter indicated with “TTAC”)was used in place of the surfactant CTAC used in Example 2 to evaluatethe stability of CFP-D. The surfactant resistance of various modifiedamadoriases was evaluated in accordance with a measurement method forsurfactant resistance according to Example 1, although under surfactanttreatment conditions in which the amadoriases were each diluted in a 20mM potassium phosphate buffer solution (pH 7.0) and mixed with 0.04%TTAC. The results are shown in Table 3-1. In this respect, it wasconfirmed that when the warmed sample was again measured for activity 30minutes after 2-fold dilution in a BSA solution, there was no change inthe activity value.

TABLE 3-1 SEQ ID NO of Residual Plasmid as Amino Acid OligonucleotideActivity Plasmid Template Enzyme Mutation Used (%) pKK223-3- None CFP-DNone None 29.2 CFP-D pKK223-3- pKK223-3- CFP-D1 E44P 23.28 43.2 CFP-D1CFP-D pKK223-3- pKK223-3- CFP-D2 E44P/E340P 16.17 69.9 CFP-D2 CFP-D1pKK223-3- pKK223-3- CFP-D3 E44P/N262H/E340P 5.6 85.3 CFP-D3 CFP-D2pKK223-3- pKK223-3- CFP-D4 E44PA/V257C/N262H/ 7.29 91.1 CFP-D4 CFP-D3E340P pKK223-3- pKK223-3- CFP-D5 E44P/E253K/V257C/ 30.31 94.9 CFP-D5CFP-D4 N262H/E340P pKK223-3- pKK223-3- CFP-D6 E44P/E133A/E253K/ 18.1996.4 CFP-D6 CFP-D5 V257C/N262H/E340P pKK223-3- pKK223-3- CFP-D7E44P/E133A/E253K/ 32.33 100 CFP-D7 CFP-D6 V257C/N262H/Q337K/ E340P

As shown in Table 3-1, the residual activity of CFP-D was 29.2% underthe conditions of this Example.

In contrast, all of the multiple variants prepared in Example 2 hadsignificantly enhanced residual activities. More specifically, theresidual activity of the double variant in which glutamic acid atposition 44 was substituted with proline and glutamic acid at position340 was substituted with proline was 69.9% and was enhanced compared tothat of CFP-D. The residual activity of the triple variant in whichasparagine at position 262 was substitutes by histidine in addition tothe prior mutation was 85.3% and was further enhanced compared to thatof CFP-D. The residual activity of the quadruple variant in which valineat position 257 was substituted with cysteine in addition to the priormutation was 91.1% and significantly enhanced compared to that of CFP-D.The residual activity of the quintuple variant in which glutamic acid atposition 253 was substituted with lysine in addition to the priormutation was 94.9%; the residual activity of the sextuple variant inwhich glutamic acid at position 133 was substituted with alanine inaddition to the prior mutation was 96.4%; and the residual activity ofthe septuple variant in which glutamine at position 337 was substitutedwith lysine in addition to the prior mutation was 100% and wassignificantly enhanced compared to CFP-D, and there was almost noinactivation of the amadoriase due to TTAC.

Thus, these amino acid substitutions were shown to enhance theresistance of the amadoriases to TTAC.

Example 3-2 (Purification of CFP-T7, CFP-D2, and CFP-D7)

Crude enzyme solutions prepared using the crude enzymes CFP-T7, CFP-D2,and CFP-D7 obtained in Examples 1 and 2 were each adsorbed to 4 ml of QSepharose Fast Flow resin (from GE Healthcare Co., Ltd.) equilibrated ina 20 mM potassium phosphate buffer solution (pH 8.0); the resin was thenwashed with 80 ml of the same buffer solution; the protein adsorbed tothe resin was subsequently eluted using a 20 mM potassium phosphatebuffer solution containing 100 mM NaCl (pH 8.0); and a fraction showingamadoriase activity was recovered.

The obtained fractions exhibiting amadoriase activity were concentratedusing Amicon Ultra-15, 30K NMWL (from Millipore Co., Ltd.). Then, theconcentrates were applied to HiLoad 26/60 Superdex 200 pg (from GEHealthcare Co., Ltd.) equilibrated in a 20 mM potassium phosphate buffersolution containing 150 mM NaCl (pH 7.0) for elution with the samebuffer solution to recover fractions showing amadoriase activity toprovide purified preparations of the wild-type and modified amadoriases.SDS-PAGE analysis confirmed that the resultant purified preparations hadbeen purified to single bands.

(Evaluation of for Various Surfactants)

Using various surfactants, the stability of the purified enzymes CFP-T7,CFP-D2, and CFP-D7 obtained as described above was evaluated. Thesurfactant resistance of the modified amadoriases was evaluated inaccordance with a measurement method for surfactant resistance accordingto Example 1 although under surfactant treatment conditions in which theamadoriases were each diluted in a 20 mM potassium phosphate buffersolution (pH 7.0) and mixed with any of various concentrations of thesurfactants. The results are shown in Table 3-2. In this respect, it wasconfirmed that when the warmed sample was again measured for activity 30minutes after 2-fold dilution in a BSA solution, there was no change inthe activity value.

TABLE 3-2 Abbreviated Carbon Addition Residual Activity(%) Name ofSurfactant Name Chain Concentration CFP-T7 D2 D7 OctyltrimethylammoniumBromide OTAB 8 0.80% 87.7 93.8 101.5 Dodecyltrimethylammonium BromideDTAB 12 0.30% 12.2 78.1 106.1 Tetradecyltrimethylammonium Bromide TTAB14 0.04% 31.7 103.1  96.6 Octadecyltrimethylammonium Bromide STAB 180.01% 59.7 99.2 94.5 Octyltrimethylammonium Chloride OTAC 8 1.20% 94.1 —100.0 Tetradecyltrimethylammonium Chloride TTAC 14 0.04% 4.1 69.9 100.0Hexadecyltrimethylammonium Chloride CTAC 16 0.04% 2.3 37.1 100.0Octadecyltrimethylammonium Chloride STAC 18 0.02% 49.3 96.1 85.2Hexadecylpyridinium Bromide 1-CPB 16 0.04% 0.7 39.6 95.3Dodecylpyridinium Chloride 1-DPC 12 0.16% 3.4 92.2 109.2Hexadecylpyridinium Chloride 1-CPC 16 0.04% 1.4 32.4 93.1N-Cetyl-4-methylpyridinium Chloride 4Me-1-CPC 16 0.04% 1.5 32.5 96.9Benzyldodecyldimethyl BDDAB 12 0.10% 1.4 94.1 105.5 ammonium BromideBenzyltetradecyldimethyl BDTAC 14 0.04% 0.7 12.1 80.0 ammonium ChlorideBenzylcetyldimethyl BCDAC 16 0.04% 1.4 76.8 88.3 ammonium ChlorideTributylhexadecyl TBCPB 16 0.04% 3.1 83.1 76.7 phosphonium Bromide

As shown in Table 3-2, the activity of CFP-T7 before introducingmutation was drastically reduced by most surfactants except when OTABand OTAC were used as surfactants. In contrast, the double variantCFP-D2 had more excellent surfactant resistance to all types of thesurfactants tested than CFP-T7. The septuple variant CFP-D7 also hadmore excellent surfactant resistance to all types of the surfactantstested than CFT-T7, and, in most cases, had enhanced surfactantresistance compared to that of the double variant CFP-D2.

As shown in Table 3-2, when OTAB (C8), DTAB (C12), TTAB (C14), and STAB(C18) having carbon chains different in length were used as surfactants,D2 as well as D7 had enhanced surfactant resistance. As such, it isbelieved that the same applies for surfactants, such asdecyltrimethylammonium bromide, whose carbon chain has 10 carbon atoms,and hexadecyltrimethylammonium bromide, whose carbon chain has 16 carbonatoms. The same also applies for OTAC (C8), TTAC (C14), CTAC (C16), andSTAC (C18) as chlorides corresponding to the bromides, and theamadoriases of the present invention are believed to have resistance todecyltrimethylammonium chloride, whose carbon chain has 10 carbon atoms,and dodecyltrimethylammonium chloride, whose carbon chain has 12 carbonatoms.

As shown in Table 3-2, both D2 and D7 had surfactant resistance, whetherthe counter ion (Z) was a chloride ion or bromide ion.

As shown in Table 3-2, both D2 and D7 had resistance to not onlyammonium ion surfactants but also pyridinium ion surfactants andphosphonium ion surfactants, showing that the surfactant resistance wasagainst cationic surfactants.

Summarizing the above results, the surfactant-resistant amadoriase ofthe present invention was demonstrated to have a wide surfactantresistance spectrum irrespective of the type of the counter ion of asurfactant, irrespective of chain length, and irrespective of the basicstructure of a cationic surfactant.

Summarizing the names and structures of the surfactants used is asfollows.

TABLE 3-3 Surfactant R1 R2 R3 R4 Structure OctyltrimethylammoniumBromide 8 1 1 1 Formula Dodecyltrimethylammonium Bromide 12 1 1 1 ITetradecyltrimethylammonium Bromide 14 1 1 1 OctadecyltrimethylammoniumBromide 18 1 1 1 Octyltrimethylammonium Chloride 8 1 1 1Tetradecyltrimethylammonium Chloride 14 1 1 1 HexadecyltrimethylammoniumChloride 16 1 1 1 Octadecyltrimethylammonium Chloride 18 1 1 1Surfactant R5 R^(a1) n¹ R^(a2) n² Structure Hexadecylpyridinium Bromide16 H 5 — — Formula Dodecylpyridinium Chloride 12 H 5 — — IIHexadecylpyridinium Chloride 16 H 5 — — N-cetyl-4-methylpyridiniumChloride 16 H 4 1 1 Surfactant R1 R2 R3 R4 StructureBenzyldodecyldimethyl- 12 1 Bn 1 Formula ammonium Bromide IBenzyltetradecyldimethyl- 14 1 Bn 1 ammonium ChlorideBenzylcetyldimethyl- 16 1 Bn 1 ammonium Chloride Surfactant R6 R7 R8 R9Structure Tributylhexadecyl- 16 4 4 4 Formula phosphonium Bromide III Hrepresents a hydrogen atom and Bn represents a benzyl group. The numeralcharacter indicates the carbon chain length of the alkyl group.

Example 4 (Evaluation for Surfactant SDS)

The stability of CFP-D was evaluated using sodium dodecyl sulfate(hereinafter indicated with “SDS”) in place of the surfactant CTAC usedin Example 2. The surfactant resistance of the modified amadoriases wasevaluated in accordance with a measurement method for surfactantresistance according to Example 1 although under surfactant treatmentconditions in which the amadoriases were each diluted in a 30 mM MES/21mM Tris buffer solution (pH 6.5) and mixed with 0.04% SDS. The resultsare shown in Table 4. In this respect, it was confirmed that when thewarmed sample was again measured for activity 30 minutes after 2-folddilution in a BSA solution, there was no change in the activity value.

TABLE 4 SEQ ID NO of Residual Plasmid as Amino Acid OligonucleotideActivity Plasmid Template Enzyme Mutation Used (%) pKK223-3- None CFP-T7None None 2.76 CFP-T7 pKK223-3- None CFP-D None None 11.3 CFP-DpKK223-3- pKK223-3- CFP-D1 E44P 23.28 11.1 CFP-D1 CFP-D pKK223-3-pKK223-3- CFP-D2 E44P/E340P 16.17 19.2 CFP-D2 CFP-D1 pKK223-3- pKK223-3-CFP-D3 E44P/N262H/E340P 5.6 11.3 CFP-D3 CFP-D2 pKK223-3- pKK223-3-CFP-D4 E44P/V257C/N262H/ 7.29 17.1 CFP-D4 CFP-D3 E340P pKK223-3-pKK223-3- CFP-D5 E44P/E253K/V257C/ 30.31 5.07 CFP-D5 CFP-D4 N262H/E340PpKK223-3- pKK223-3- CFP-D6 E44P/E133A/E253K/ 18.19 3.62 CFP-D6 CFP-D5V257C/N262H/E340P pKK223-3- pKK223-3- CFP-D7 E44P/E133A/E253K/ 32.333.92 CFP-D7 CFP-D6 V257C/N262H/Q337K/ E340P

As shown in Table 4, the residual activity of CFP-T7 was 2.76% under theconditions of this Example.

In contrast, all of the multiple variants prepared in Example 2 hadsignificantly enhanced residual activities. More specifically, theresidual activity of the double variant in which glutamic acid atposition 44 was substituted with proline and glutamic acid at position340 was substituted with proline was 19.2% and was enhanced compared tothat of CFP-T7. The residual activity of the triple variant in whichasparagine at position 262 was substitutes by histidine in addition tothe prior mutation was 11.3% and was further enhanced compared to thatof CFP-T7. The residual activity of the quadruple variant in whichvaline at position 257 was substituted with cysteine in addition to theprior mutation was 17.1% and enhanced compared to that of CFP-T7. Theresidual activity of the quintuple variant in which glutamic acid atposition 253 was substituted with lysine in addition to the priormutation was 5.07%; the residual activity of the sextuple variant inwhich glutamic acid at position 133 was substituted with alanine inaddition to the prior mutation was 3.62%; the residual activity of theseptuple variant in which glutamine at position 337 was substituted withlysine in addition to the prior mutation was 3.92% and was enhancedcompared to CFP-T7.

Thus, these amino acid substitutions were shown to enhance theresistance of the amadoriases to SDS.

Example 5 (Measurement of Fructosyl Peptide Sample Under Mixing ofSurfactant)

The purified enzyme of CFP-T7 and CFP-D7 obtained in Example 3-2 wasused to measure a sample as shown below. The activity values of CFP-T7and CFP-D7 were determined using (reagent 1) adjusted to pH 7.0 withαFVH at a final concentration of 5 mM as a substrate according to themethod for measuring amadoriase activity.

(11) Preparation of Fructosyl Peptide Sample

(Reagent 4) αFVH (125 mg) was dissolved in ion-exchanged water, thevolume of the solution was fixed to 10 ml, and thereby a 30 mM substratesolution was obtained. In addition, the resultant was diluted by 1/714with a CTAC solution to provide a 42 μM αFVH/0% to 0.2% CTAC solution.

(12) Measurement of Fructosyl Peptide Sample

(Reagent 5)

0.21 mM DA-64 sodium(N-(Carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylamine,from Wako Pure Chemical Industries Ltd.

20 mM Potassium phosphate buffer solution (pH 7.0)

(Reagent 6)

6.7 U/ml CFP-T7 or CFP-D7

19 U/ml Peroxidase (from Kikkoman Corporation)

20 mM Potassium phosphate buffer solution (pH 7.0)

(Reagent 6) (50 μl) was added to a mixed solution of 135 μl of (reagent5) warmed at 37° C. for 5 minutes in advance and 25 μl of the sampleprepared in (11) above to start reaction, and absorbance at a wavelengthof 751 nm after reaction at 37° C. for 5 minutes was measured using anautomated analyzer, Bio Majesty JCA-BM1650 (from JEOL Ltd.). Absorbance(reagent blank) at a wavelength of 751 nm measured by a similaroperation for (reagent 4) prepared using ion-exchanged water in place ofthe substrate solution was used as a control to calculate the amount ofchange in absorbance (difference) when each sample was measured, usingthe following equation. The final concentration of a colorimetricsubstrate, DA-64, was 0.15 mM; the final concentration of αFVH in thecase of the presence of a substrate was 5 μM; and the length (opticalpath) of the cell used for absorbance measurement was 1 cm.

Amount of change in absorbance=ΔAes−Ae0

ΔAes: absorbance after a lapse of 5 minutes from reaction initiation

Ae0: absorbance after a lapse of 5 minutes from reaction initiation whenthe control solution was added

The amount of change in absorbance when αFVH/0% to 0.2% CTAC were usedas samples was calculated. The results are shown in Table 5.

TABLE 5 Amount of Change in Absorbance at Wavelength of 751 nm underMixing of Each Concentration of CTAC Mixing of CTAC (%) 0 0.005 0.010.02 0.04 0.06 0.08 0.1 0.14 0.2 Enzyme CFP-T7 0.150 0.111 0.077 0.0310.005 (Not carried out) Enzyme CFP-D7 0.150 0.152 0.147 0.140 0.1310.091 0.083 0.073 0.068 0.069

As shown in Table 5, under the conditions of this Example, the amount ofchange in the absorbance of CFP-T7 under the mixing of 0% CTAC was 0.150and the amount of change in the absorbance of CFP-T7 under the mixing of0.005% CTAC was 0.111. In addition, the amount of change in theabsorbance of CFP-T7 under the mixing of 0.01% CTAC was 0.077; theamount of change in the absorbance of CFP-T7 under the mixing of 0.02%CTAC was 0.031; and the amount of change in the absorbance of CFP-T7under the mixing of 0.04% CTAC decreased to 0.005, showing that a higherconcentration of CTAC decreased the amount of change in absorbance.

In contrast, the amount of change in the absorbance of CFP-D7 under themixing of 0.02% CTAC was 0.140 and the amount of change in theabsorbance of CFP-D7 under the mixing of 0.2% CTAC was 0.069. That is,whereas the mixing of more CTAC decreased the amount of change in theabsorbance of CFP-T7, it suppressed a decrease in the amount of changein the absorbance of CFP-D7; the presence of 0.1% or more CTAC made theamount of change in the absorbance of CFP-D7 constant. The diminution inthe amount of change in absorbance was large until the CTACconcentration reached 1.3 mM (0.04%) as the critical micelleconcentration thereof; however, the concentration exceeding the criticalmicelle concentration decreased a change in effect on the amadoriase.Thus, CFP-D7 was found to be stably present under the mixing of a highconcentration of CTAC, enabling the measurement of αFVH.

Example 6 (Quantification of Fructosyl Peptide Sample Under Mixing ofSurfactant)

Using the purified enzymes of CFP-T7 and CFP-D7, the linearity of αFVHmeasurement values was compared in the range of 0.5 to 3 μM under themixing of CTAC. For CFP-T7, the amount of change in absorbance wasmeasured as in Example 5 under conditions of mixing 0.01% or 0.02% CTACand further using αFVH at 4.2 μM, 8.4 μM, 13 μM, 17 μM, 21 μM, or 25 μM,i.e., at a final concentration of 0.5 μM, 1.0 μM, 1.5 μM, 2.0 μM, 2.5μM, or 3.0 μM to calculate a correlation coefficient. Similarly, forCFP-D7, the amount of change in absorbance was measured as in Example 5under conditions of mixing 0.02% or 0.2% CTAC and using the sameconcentrations of αFVH to calculate a correlation coefficient. Theresults are shown in Table 6, and the correlation data are shown inFIGS. 2, 3, 4, and 5. FIG. 2 shows the results of measuring αFVH usingCFP-T7 under the mixing of 0.01% CTAC; FIG. 3 shows the results ofmeasuring αFVH using CFP-T7 under the mixing of 0.02% CTAC; FIG. 4 showsthe results of measuring αFVH using CFP-D7 under the mixing of 0.02%CTAC; and FIG. 5 shows the results of measuring αFVH using CFP-D7 underthe mixing of 0.2% CTAC.

TABLE 6 Correlation Coefficient under Mixing of Each Concentration ofCTAC Mixing of CTAC (%) 0.01  0.02 0.2  Enzyme CFP-T7 0.924 0.675 (Notcarried out) Enzyme CFP-D7 (Not carried out) 0.985 0.965

As shown in Table 6, under the conditions of this Example, thecorrelation coefficient of 0.5 μM to 3.0 μM αFVH for CFP-T7 was as highas 0.924 under the mixing of 0.01% CTAC but as low as 0.625 under themixing 0.02% CTAC. In contrast, when CFP-D7 was used, the correlationcoefficient of 0.5 μM to 3.0 μM αFVH indicated a linearity as high as0.965 even under the mixing of 0.2% CTAC.

According to the package insert of sank HbAlc (from Ark Ray Inc.) as aHbAlc measurement kit for an enzyme method, a whole blood specimen isreacted in a state diluted by 1/416 with an amadoriase. For example,when the HbAlc with an NGSP value is 6.5%, the whole Hb concentration is141 to 150 g/l, and the blood sample is measured by dilution by 1/416,the concentration of αFVH excised by protease is 0.50 to 0.53 μM. Theactual border line of the HbAlc value for diabetic diagnosis is 6.5%(NGSP). Thus, CFP-D7 can be sufficiently used in the actual measurementof HbAlc, and can be said to be capable of being used in combinationwith, for example, CTAC to increase measurement sensitivity.

Example 7 (Evaluation of Surfactant Resistance of Various Amadoriases)

To provide a composition for measuring glycated hemoglobin, containingan amadoriase capable of having activity remaining even in the presenceof a surfactant, preferably an ionic surfactant, an amadoriase derivedfrom the genus Coniochaeta was modified as described above to enhancethe surfactant resistance thereof. It is not known whether or not HbAlccan be measured in the presence of an ionic surfactant for an amadoriaseother than the amadoriase derived from the genus Coniochaeta.Accordingly, the measurement of αFVH was attempted by combining afructosyl peptide oxidase derived from Phaeosphaeria nodorum or aketoamine oxidase derived from Neocosmospora vasinfecta with an ionicsurfactant.

1. Production and Purification of Fructosyl Peptide Oxidase Derived fromPhaeosphaeria nodorum

SEQ ID NO: 40 shows the amino acid sequence of fructosyl peptide oxidasederived from Phaeosphaeria nodorum (hereafter referred to as “PnFX”)(see Biotechnology and Bioengineering, 106, 358-366, 2010). The gene(SEQ ID NO: 44) encoding the amino acid sequence as shown in SEQ ID NO:40 was obtained via total synthesis of cDNA by a conventional techniqueof PCR of a gene fragment. The NdeI site and the BamHI were added to the5′ terminus and the 3′ terminus of SEQ ID NO: 40, respectively. Further,the full-length amino acid sequence that is deduced based on the clonedgene sequence was confirmed to be consistent with the PnFX sequence asshown in FIG. 1. Subsequently, in order to express the gene shown in SEQID NO: 44 in E. coli, the following procedures were performed. The genefully synthesized above was treated with two types of restrictionenzymes, NdeI and BamHI (manufactured by Takara Bio Inc.) and insertedinto the NdeI-BamHI site of the pET-22b(+) vector (manufactured byNovagen, Inc.). Thus, the recombinant plasmid pET22b-PnFX was obtained.Strains of E. coli BL21 (DE3) were transformed under the conditions asdescribed above to obtain a strain of E. coli (DE3) (pET22b-PnFX).

The strains of E. coli BL21 (DE3) (pET22b-PnFX) capable of producingPnFX obtained in the manner described above were inoculated into LB-ampmedia supplemented with IPTG (final concentration: 0.1 mM) and culturedtherein at 25° C. for 16 hours. The resulting cultured strains werewashed with a 10 mM potassium phosphate buffer (pH 8.0), the washedstrains were suspended in the same buffer, the resulting suspension wasultrasonically disintegrated, and the resultant was centrifuged at20,000×g for 10 minutes to prepare a crude enzyme solution.

The prepared crude enzyme solution containing PnFX was purified inaccordance with the method described in the non-patent document(Biotechnology and Bioengineering, 106, 358-366, 2010). Morespecifically, the crude enzyme solution was fractionated with ammoniumsulfate, dialyzed against a 10 mM potassium phosphate buffer (pH8.0),purified via anion-exchange chromatography (Q Sepharose Fast Flow wasused in Example 2), and then purified via gel filtration chromatography(HiLoad 26/600 Sueprdex 200 was used in Example 2). The obtainedfraction was analyzed by SDS-PAGE to confirm that the fraction wassufficiently purified, so that no other contaminating proteins werepresent therein. The fraction was designated to be a purified sample ofPnFX.

2. Production and Purification of Ketoamine Oxidase Derived fromNeocosmospora vasinfectaSEQ ID NO: 38 is the amino acid sequence of a ketoamine oxidase derivedfrom Neocosmospora vasinfecta (NvFX), and the activity of NvFX has beenidentified by expressing a recombinant plasmid, pET22b-NvFX, into whichthe gene (SEQ ID NO: 45) encoding the amino acid sequence of SEQ ID NO:38 is inserted, in Escherichia coli (see International Publication No.WO 2012/18094). Escherichia coli strain BL21 (DE3) was transformed as inExample 1, and the obtained Escherichia coli strain BL21 (DE3)(pET22b-NvFX) was used and cultured by the above method and a crudeenzyme solution of NvFX was prepared.

The prepared crude enzyme solution was allowed to adsorb to Q SepharoseFast Flow resin (manufactured by GE Healthcare) equilibrated with a 10mM potassium phosphate buffer (pH8.0), the resin was washed with a 10 mMpotassium phosphate buffer (pH8.0) containing 20 mM NaCl, and NvFXadsorbed to the resin was then eluted and collected with the aid of a 10mM potassium phosphate buffer (pH8.0) containing 300 mM NaCl.

The obtained crude enzyme solution of NvFX was applied to HiLoad 26/60Superdex 200 column equilibrated in a 20 mM MES-NaOH buffer solutioncontaining 150 mM NaCl (pH 7.0) to elute NvFX with the same buffersolution to recover a fraction showing fructosyl amino acid oxidaseactivity (amadoriase activity). The resultant fraction was analyzed bySDS-PAGE to confirm that the fraction was sufficiently purified, so thatno other contaminating proteins were present therein, and was used as apurified preparation of NvFX.

The purified preparations of amadoriases obtained as described abovewere used as samples to measure αFVH under the mixing of CTAC using PnFXand NvFX as in Example 5. The results are shown in Table 7.

TABLE 7 Amount of Change in Absorbance at Wavelength of 751 nm underMixing of Each Concentration of CTAC Mixing of CTAC (%) 0 0.01 0.02 0.040.06 0.1 0.2 Enzyme CFP-T7 0.150 0.077 0.031 0.005 (Not carried out)Enzyme PnFX 0.173 0.115 0.084 0.026 0.008 0.003 0.001 Enzyme NvFX 0.1180.013 0.013 0.007 0.004 0.003 0.001

As shown in Table 7, under the conditions of this Example, the amount ofchange in the absorbance of PnFX under the mixing of 0% CTAC was 0.173and the amount of change in the absorbance under the mixing of 0.01%CTAC was 0.115. The amount of change in the absorbance of PnFX under themixing of 0.02% CTAC was 0.084, whereas the amount of change in theabsorbance of CFP-T7 under the mixing thereof was 0.031; and the amountof change in the absorbance of PnFX under the mixing of 0.04% CTAC was0.026, whereas the amount of change in the absorbance of CFP-T7 underthe mixing thereof was 0.005. Thus, PnFX is capable of measuring αFVH asa substrate under the mixing of CTAC at a concentration as high as 0.02%or more.

For NvFX, absorbance was increased under the mixing of 0.02% or lessCTAC; however, αFVH could not accurately be measured since the influenceof the occurrence of turbidity increased absorbance even in the blankusing ion-exchanged water in place of the substrate.

Example 8 (Evaluation of Surfactant Resistance of Amadoriase in Presenceof any of Buffering Agents)

It was studied whether the surfactant resistance of an amadoriase isenhanced or not when a buffering agent other than a 30 mM MES/21 mM Trisbuffering agent (pH 6.5) was used. Using CFP-T7 purified as describedabove as a sample, the final concentration of CTAC was set at 0.01% toevaluate the surfactant resistance of CFP-T7 according to themeasurement method for surfactant resistance in Example 1 in thepresence of any of various buffering agents, specifically a phosphatebuffering agent containing phosphoric acid and potassium phosphate (pH7.0), a citrate buffering agent containing citric acid and sodiumcitrate (pH 6.0), an MES buffering agent containing MES and it sodiumsalt (pH 7.0), an HEPES buffering agent containing HEPES and its sodiumsalt (pH 7.0), and an ACES buffering agent containing ACES and itssodium salt (pH 7.0), in place of the 30 mM MES/21 mM Tris bufferingagent (pH 6.5). The results are shown in Table 8. In this respect, itwas confirmed that when the warmed sample was again measured foractivity 30 minutes after 2-fold dilution in a BSA solution, there wasno change in the activity value.

TABLE 8 CFP-T7 Residual Activity Buffer Solution Concentration (%)Phosphate 20 mM 15.1 50 mM 27.2 100 mM 68.3 150 mM 88.5 300 mM 98.4Citrate 10 mM 104.1 50 mM 108.2 100 mM 105.7 MES 50 mM 14.3 100 mM 17.6150 mM 62.9 300 mM 95.8 HEPES 50 mM 13.2 100 mM 13.2 150 mM 12.2 300 mM9.6 ACES 20 mM 15.6 100 mM 16.4 200 mM 22.1

As shown in Table 8, under the conditions of this Example, thesurfactant resistance of CFP-T7 was demonstrated to be enhanced in amanner dependent on the concentration of a buffering agent in thepresence of the phosphate buffering agent, the MES buffering agent, orthe ACES buffering agent. The citrate buffering agent was particularlyuseful since inactivation due to the surfactant did not occur even at 10mM of this agent. The effect of maintaining amadoriase activity was notobserved for the HEPES buffering agent. The above results show,surprisingly, that the phosphate buffering agent, the citrate bufferingagent, the MES buffering agent, and the ACES buffering agent have theeffect of enhancing the surfactant resistance of the amadoriase.

Example 9 (Evaluation of Surfactant Resistance of Amadoriase DuringAddition of Each Stabilizer)

It was studied whether or not the addition of any of various stabilizersenhanced the surfactant resistance of an amadoriase. Phosphoric acid, atricarboxylic acid (e.g., citric acid), a dicarboxylic acid (e.g., malicacid, maleic acid, citraconic acid, malonic acid, glutaric acid, ortartaric acid), a monpcarboxylic acid (e.g., acetic acid), MES, MOPS,MOPSO, or ammonium sulfate was used as a stabilizer. As a ComparativeExample, CHES was used. To prevent change in pH when adding thestabilizer, 500 mM HEPES (pH 7.0) was used as a buffer solution; CFP-T7purified as described above was used as a sample; the finalconcentration of CTAC was set at 0.01%; and any of the stabilizers wasfurther added to evaluate the surfactant resistance of CFP-T7 accordingto the measurement method for surfactant resistance in Example 1. Theresults are shown in Tables 9-1 and 9-2. Using a 500 mM HEPES (pH 7.0)buffering agent and CFP-D2 purified as described above as a sample, anyof the stabilizers was further added, and the surfactant resistance ofCFP-D2 was evaluated according to the measurement method for surfactantresistance in Example 1 although under more harsh surfactant treatmentconditions in which the final concentration of CTAC was set at 0.08% andthe treatment temperature at 37° C. The results are shown in Table 9-3.It was confirmed that the pH actually indicated 7.0 when the stabilizerwas added. Incidentally, it was confirmed that when the warmed samplewas again measured for activity 30 minutes after 2-fold dilution in aBSA solution, there was no change in the activity value.

TABLE 9-1 CFP-T7 Residual Activity Stabilizer Concentration (%) None —4.7 Phosphate 2 mM 28.1 5 mM 61.6 10 mM 76.8 20 mM 98.6 50 mM 100.0Citrate 0.2 mM 12.2 0.5 mM 30.7 2 mM 87.6 Malate 2 mM 12.6 5 mM 35.4 10mM 74.3 Acetate 10 mM 8.9 20 mM 20.7 50 mM 52.4 MES 10 mM 19.5 20 mM54.1 40 mM 103.0 Ammonium Sulfate 2 mM 32.7 5 mM 82.3 10 mM 94.2

TABLE 9-2 CFP-T7 Residual Activity Stabilizer Concentration (%) None —3.1 Maleate 2 mM 15.5 10 mM 81.5 Citraconate 2 mM 18.5 10 mM 89.1Malonate 2 mM 10.8 10 mM 77.3 Glutarate 2 mM 4.5 10 mM 55.9 Tartrate 2mM 5.3 10 mM 70.0 MOPS 10 mM 6.5 20 mM 20.6 MOPSO 10 mM 9.0 20 mM 23.5CHES 20 mM 3.0

TABLE 9-3 CFP-D2 Residual Activity Stabilizer Concentration (%) None —9.4 Phosphate 5 mM 72.8 Malate 5 mM 59.2 MOPS 20 mM  42.4

Tables 9-1 and 9-2 demonstrated that under the conditions of thisExample, the addition of phosphoric acid, citric acid, malic acid,maleic acid, citraconic acid, malonic acid, glutaric acid, tartaricacid, acetic acid, MES, MOPS, MOPSO, or ammonium sulfate as a stabilizerenhanced the surfactant resistance of CFP-T7 in a manner dependent onthe concentration of the stabilizer. In particular, citric acid wasfound to be capable of enhancing the surface resistance of CFP-T7 evenwhen added at a trace amount of 0.2 mM. As shown in Table 9-3, theaddition of phosphoric acid, malic acid, or MOPS as a stabilizersignificantly enhanced the surfactant resistance of the purified CFP-D2enzyme compared to when the stabilizer is absent. It was not known thatthe stability of an amadoriase to a surfactant can be enhanced byvarious compounds and this was surprising. In particular, CHES havingthe following structure:

did not have any stabilizing action, whereas compounds included informula (IV)

[wherein, n may be 0, 1, 2 or 3; each R¹⁰ independently represent H, OH,—CH₂OH or —COOH], whose structures are highly analogous to the abovestructure, MES (n=1 and R¹⁰ represents H), MOPS (n=2 and R¹⁰ eachrepresent H), MOPSO (n=2 and a plurality of R¹⁰ each represent OH or H),surprisingly, had an amadoriase-stabilizing action. The above resultsshow that phosphoric acid, tricarboxylic acids, dicarboxylic acids,monocarboxylic acids, and compounds represented by formula (IV), such asMES, MOPS, and MOPSO, have the effect of enhancing the surfactantresistance of an amadoriase. Further, enhanced surfactant resistance wasobserved regarding CFP-T7, the amadoriase to which the mutation of thepresent invention was not introduced, as well as regarding CFP-D2, theamadoriase to which the mutation of the present invention wasintroduced.

The combination of the results of Tables 8 and 9 shows that theamadoriase-stabilizing action of the stabilizer of the present inventionis an action different from the amadoriase-stabilizing action of thebuffering agent of the present invention. More specifically, it wasconfirmed from Table 8 that the use of MES at a concentration of 50 mMas a buffering agent of the present invention resulted in a residualactivity of CFP-T7 amadoriase of 14.3%, the use thereof at aconcentration of 100 mM resulted in a residual activity of the enzyme of17.6%, and the use thereof at a concentration of 150 mM resulted in aresidual activity of the enzyme of 62.9%. In contrast, it was confirmedfrom Table 9 that the use of MES at a concentration of 10 mM as astabilizer of the present invention while using HEPES (pH 7.0) merely asa pH buffer agent having no amadoriase-stabilizing action resulted in aresidual activity of CFP-T7 amadoriase of 19.5% and the use of MES at aconcentration of 20 mM resulted in a residual activity of the enzyme of54.1%. In other words, MES exhibited an amadoriase-stabilizing action ata concentration as low as 20 mM incapable of sufficiently exerting abuffer capacity, and the confirmed residual activity of the amadoriasesurprisingly exceeded the residual activity (Table 8, 17.6%) when MESwas used at a concentration of 100 mM as a buffering agent of thepresent invention. Thus, the amadoriase-stabilizing action of thestabilizer of the present invention is a stabilizing action differentfrom the amadoriase-stabilizing action of the buffering agent of thepresent invention. The same applies to phosphoric acid and citric acid.It is believed that the same applies to dicarboxylic acids, MOPS, andMOPSO exhibiting a stabilizing action since these can also be used asbuffering agents.

(Evaluation of Surfactant Resistance of PnFX During Addition of EachStabilizer)

It was studied whether or not the above stabilizers had a surfactantresistance-enhancing effect on amadoriases other than the amadoriasederived from the genus Coniochaeta, for example, PnFX. The samestabilizers as those described above were used as stabilizers; 300 mMHEPES (pH 7.0) was used as a buffering agent to prevent a change in pHwhen the stabilizers was each added; PnFX purified as described abovewas used as a sample; the final concentration of CTAC was set at 0.04%;and the surfactant resistance of PnFX was evaluated as described above.It was confirmed that the pH actually indicated 7.0 when a stabilizerwas added. The results are shown in Table 10. In this respect, it wasconfirmed that when the warmed sample was again measured for activity 30minutes after 2-fold dilution in a BSA solution, there was no change inthe activity value.

TABLE 10 PnFX Residual Activity Stabilizer Concentration (%) None — 27.9Phosphate 5 mM 37.5 Citrate 0.5 mM 47.3 Malate 5 mM 60.4 Acetate 20 mM42.6 MES 20 mM 89.6 Ammonium Sulfate 5 mM 53.0

As shown in Table 10, under the conditions of this Example, the additionof phosphoric acid, citric acid, malic acid, acetic acid, MES, orammonium sulfate had a surfactant resistance-enhancing effect on PnFX aswith CFP-T7. Thus, phosphoric acid, tricarboxylic acids, dicarboxylicacids, monocarboxylic acids, MES, and ammonium sulfate are useful asstabilizers for enhancing the surfactant resistance of a wide variety ofamadoriases. Since CFP-T7 has 75% amino acid sequence identity to PnFX,amadoriases having 75% amino acid sequence identity to CFP-T7 can besaid to have the above effect.

SEQUENCE LISTING FREE TEXT

SEQ ID NO: 1. Amino Acid Sequence of CFP-T7

SEQ ID NO: 2. Gene Sequence for CFP-T7

SEQ ID NO: 3. Amino Acid Sequence of CFP-D

SEQ ID NO: 4. Gene Sequence for CFP-D

SEQ ID NO: 5. N262H Introducing Primer Fw

SEQ ID NO: 6. N262X Introducing Primer Rv

SEQ ID NO: 7. V257C Introducing Primer Fw

SEQ ID NO: 8. V257X Introducing Primer Rv

SEQ ID NO: 9. V257S Introducing Primer Fw

SEQ ID NO: 10. V257T Introducing Primer Fw

SEQ ID NO: 11. E253K Introducing Primer Fw

SEQ ID NO: 12. E253X Introducing Primer Rv

SEQ ID NO: 13. E253R Introducing Primer Fw

SEQ ID NO: 14. Q337K Introducing Primer Fw

SEQ ID NO: 15. Q337X Introducing Primer Rv

SEQ ID NO: 16. E340P Introducing Primer Fw

SEQ ID NO: 17. E340X Introducing Primer Rv

SEQ ID NO: 18. E133A Introducing Primer Fw

SEQ ID NO: 19. E133X Introducing Primer Rv

SEQ ID NO: 20. E133M Introducing Primer Fw

SEQ ID NO: 21. E133K Introducing Primer Fw

SEQ ID NO: 22. E44P Introducing Primer Fw

SEQ ID NO: 23. E44X Introducing Primer Rv

SEQ ID NO: 24. G256K Introducing Primer Fw

SEQ ID NO: 25. G256R Introducing Primer Fw

SEQ ID NO: 26. E81K Introducing Primer Fw

SEQ ID NO: 27. E81X Introducing Primer Rv

SEQ ID NO: 28. F43Y/E44P Introducing Primer Fw

SEQ ID NO: 29. V257X/N262H Introducing Primer Rv

SEQ ID NO: 30. E253K/V257C Introducing Primer Fw

SEQ ID NO: 31. E253X/V257C/N262H Introducing Primer Rv

SEQ ID NO: 32. Q337K/E340P Introducing Primer Fw

SEQ ID NO: 33. Q337X/E340P Introducing Primer Rv

SEQ ID NO: 34. Amadoriase Derived from Eupenicillium terrenum

SEQ ID NO: 35. Ketoamine Oxidase Derived from Pyrenochaeta sp.

SEQ ID NO: 36. Ketoamine Oxidase Derived from Arthrinium sp.

SEQ ID NO: 37. Ketoamine Oxidase Derived from Curvularia clavata

SEQ ID NO: 38. Ketoamine Oxidase Derived from Neocosmospora vasinfecta

SEQ ID NO: 39. Fructosyl Amino Acid Oxidase Derived from Cryptococcusneoformans

SEQ ID NO: 40. Fructosyl Peptide Oxidase Derived from Phaeosphaerianodorum

SEQ ID NO: 41. Fructosyl Amino Acid Oxidase Derived from Aspergillusnidulans

SEQ ID NO: 42. Fructosyl Amino Acid Oxidase Derived from Ulocladium sp.

SEQ ID NO: 43. Fructosyl Amino Acid Oxidase Derived from Penicilliumcrysogenum

SEQ ID NO: 44. Gene for Fructosyl Peptide Oxidase Derived fromPhaeosphaeria nodorum

SEQ ID NO: 45. Gene for Ketoamine Oxidase Derived from Neocosmosporavasinfecta

SEQ ID NO: 46. D129K Introducing Primer Fw

SEQ ID NO: 47. D129K Introducing Primer Rv

SEQ ID NO: 48. D132K Introducing Primer Fw

SEQ ID NO: 49. E231K Introducing Primer Fw

SEQ ID NO: 50. E231X Introducing Primer Rv

SEQ ID NO: 51. D232K Introducing Primer Fw

SEQ ID NO: 52. E249K Introducing Primer Fw

SEQ ID NO: 53. E249K Introducing Primer Rv

SEQ ID NO: 54. E249K/V257C Introducing Primer Rv

SEQ ID NO: 55. Gene for Ketoamine Oxidase Derived from Curvulariaclavata

SEQ ID NO: 56. D129K Introducing Primer Fw for CcFX

SEQ ID NO: 57. D129K Introducing Primer Rv for CcFX

SEQ ID NO: 58. D132K Introducing Primer Fw for CcFX

SEQ ID NO: 59. E133K Introducing Primer Fw for CcFX

SEQ ID NO: 60. E133A Introducing Primer Fw for CcFX

SEQ ID NO: 61. E133X Introducing Primer Rv for CcFX

SEQ ID NO: 62. E229K Introducing Primer Fw for CcFX

SEQ ID NO: 63. D230K Introducing Primer Fw for CcFX

SEQ ID NO: 64. D230X Introducing Primer Rv for CcFX

SEQ ID NO: 65. E247K Introducing Primer Fw for CcFX

SEQ ID NO: 66. E247K Introducing Primer Rv for CcFX

SEQ ID NO: 67. E251K Introducing Primer Fw for CcFX

SEQ ID NO: 68. E251X Introducing Primer Rv for CcFX

SEQ ID NO: 69. E251R Introducing Primer Fw for CcFX

SEQ ID NO: 70. N254K Introducing Primer Fw for CcFX

SEQ ID NO: 71. N254K Introducing Primer Rv for CcFX

SEQ ID NO: 72. T335K Introducing Primer Fw for CcFX

SEQ ID NO: 73. T335X Introducing Primer Rv for CcFX

SEQ ID NO: 74. T335R Introducing Primer Fw for CcFX

All publications, patents, and patent applications cited in thisapplication are intended to be incorporated herein by reference in theirentirety.

1. A method for measuring glycated hemoglobin said method comprising useof one or more surfactants and an amadoriase, wherein the amadoriasecomprises an amino acid sequence having an identity of at least 90% withthe amino acid sequence as shown in SEQ ID NO: 1, 3, 37 or 40, and (i)has a residual activity (%) of 15% or higher 5 minutes after an ionicsurfactant is added compared with a case where no ionic surfactant isadded, and/or (ii) exhibits a difference of 0.006 or higher betweenabsorbance at 751 nm after a colorimetric substrate sodiumN-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylamine(DA-64) is added and reacted for 5 minutes, and absorbance at 751 nm 5minutes after a control solution containing ion-exchanged water in placeof a glycated amino acid solution or a glycated peptide solution isadded, in the presence of a 0.04% final concentration of surfactant,wherein, when the amadoriase has an amino acid sequence having anidentity of at least 90% with the amino acid sequence as shown in SEQ IDNO: 1, 3, or 37, then the amadoriase comprises one or moresubstitution(s) selected from the group consisting of: (i) substitutionof the amino acid at the position corresponding to position 257 in SEQID NO 1 with cysteine, serine, or threonine; (ii) substitution of theamino acid at the position corresponding to position 253 in SEQ ID NO 1with lysine, or arginine; (iii) substitution of the amino acid at theposition corresponding to position 262 in SEQ ID NO 1 with histidine;(iv) substitution of the amino acid at the position corresponding toposition 337 in SEQ ID NO 1 with lysine, or arginine; (v) substitutionof the amino acid at the position corresponding to position 249 in SEQID NO 1 with lysine, or arginine; (vi) substitution of the amino acid atthe position corresponding to position 340 in SEQ ID NO 1 with proline;(vii) substitution of the amino acid at the position corresponding toposition 232 in SEQ ID NO 1 with lysine, or arginine; (viii)substitution of the amino acid at the position corresponding to position129 in SEQ ID NO 1 with lysine, or arginine; (ix) substitution of theamino acid at the position corresponding to position 132 in SEQ ID NO 1with lysine, or arginine; (x) substitution of the amino acid at theposition corresponding to position 133 in SEQ ID NO 1 with alanine,methionine, lysine, or arginine; (xi) substitution of the amino acid atthe position corresponding to position 44 in SEQ ID NO 1 with proline;(xii) substitution of the amino acid at the position corresponding toposition 256 in SEQ ID NO 1 with lysine, or arginine; (xiii)substitution of the amino acid at the position corresponding to position231 in SEQ ID NO 1 with lysine, or arginine; and (xiv) substitution ofthe amino acid at the position corresponding to position 81 in SEQ ID NO1 with lysine, or arginine; wherein, when the ionic surfactant is acationic surfactant, the substitution is: (i) substitution of the aminoacid at the position corresponding to position 257 in SEQ ID NO 1 withcysteine, serine, or threonine; (ii) substitution of the amino acid atthe position corresponding to position 253 in SEQ ID NO 1 with lysine,or arginine; (iii) substitution of the amino acid at the positioncorresponding to position 262 in SEQ ID NO 1 with histidine; (iv)substitution of the amino acid at the position corresponding to position337 in SEQ ID NO 1 with lysine, or arginine; (v) substitution of theamino acid at the position corresponding to position 249 in SEQ ID NO 1with lysine, or arginine; (vi) substitution of the amino acid at theposition corresponding to position 340 in SEQ ID NO 1 with proline;(vii) substitution of the amino acid at the position corresponding toposition 232 in SEQ ID NO 1 with lysine, or arginine; (viii)substitution of the amino acid at the position corresponding to position129 in SEQ ID NO 1 with lysine, or arginine; (ix) substitution of theamino acid at the position corresponding to position 132 in SEQ ID NO 1with lysine, or arginine; (x) substitution of the amino acid at theposition corresponding to position 133 in SEQ ID NO 1 with alanine,methionine, lysine, or arginine; (xi) substitution of the amino acid atthe position corresponding to position 44 in SEQ ID NO 1 with proline;(xii) substitution of the amino acid at the position corresponding toposition 256 in SEQ ID NO 1 with lysine, or arginine; (xiii)substitution of the amino acid at the position corresponding to position231 in SEQ ID NO 1 with lysine, or arginine; or (xiv) substitution ofthe amino acid at the position corresponding to position 81 in SEQ ID NO1 with lysine, or arginine; then said improved residual activity isimproved residual activity after a cationic surfactant is added; andwherein, when the ionic surfactant is an anionic surfactant, thesubstitution is: (i) substitution of the amino acid at the positioncorresponding to position 257 in SEQ ID NO 1 with cysteine, serine, orthreonine; or (vi) substitution of the amino acid at the positioncorresponding to position 340 in SEQ ID NO 1 with proline.
 2. The methodof claim 1, wherein the surfactant has a critical micelle concentrationof 70 mM or lower.
 3. The method of claim 2, wherein the surfactant isone or more ionic surfactants selected from the group consisting of aquaternary ammonium salt represented by the following general formula(I):

wherein, R¹ to R⁴, which may be the same or different, each represent asubstituted or unsubstituted C₁ to C₂₀ alkyl, alkenyl, aryl or benzyl;and Z⁻ represents a monovalent anion, octyltrimethylammonium chloride,octyltrimethylammonium bromide, dioctyldimethylammonium chloride,dioctyldimethylammonium bromide, decyltrimethylammonium chloride,decyltrimethylammonium bromide, dodecyltrimethylammonium chloride,dodecyltrimethylammonium bromide, tetradecyltrimethylammonium chloride,tetradecyltrimethylammonium bromide, hexadecyltrimethylammoniumchloride, hexadecyltrimethylammonium bromide, octadecyltrimethylammoniumchloride, octadecyltrimethylammonium bromide, eicosyltrimethylammoniumchloride and eicosyltrimethylammonium bromide,benzyldodecyldimethylammonium chloride, benzyldodecyldimethylammoniumbromide, benzyltetradecyldimethylammonium chloride,benzyltetradecyldimethylammonium bromide, benzylcetyldimethylammoniumchloride, and benzylcetyldimethylammonium bromide, a pyridinium saltrepresented by the following general formula (II):

wherein, R⁵ represents a substituted or unsubstituted C₁ to C₂₀ alkyl, aplurality of R^(a), which may be the same or different, each represent ahydrogen atom or a substituted or unsubstituted C₁ to C₂₀ alkyl,alkenyl, aryl or benzyl; n represents an integer of 1 to 5; and Z⁻represents a monovalent anion, 1-decylpyridinium chloride,1-decylpyridinium bromide, 1-dodecylpyridinium chloride,1-dodecylpyridinium bromide, 1-tetradecylpyridinium chloride,1-tetradecylpyridinium bromide, 1-hexadecylpyridinium chloride,1-hexadecylpyridinium bromide, N-cetyl-2-methylpyridinium chloride,N-cetyl-2-methylpyridinium bromide, N-cetyl-3-methylpyridinium chloride,N-cetyl-3-methylpyridinium bromide, N-cetyl-4-methylpyridinium chloride,N-cetyl-4-methylpyridinium bromide, 1-octadecylpyridinium chloride,1-octadecylpyridinium bromide, 1-eicosylpyridinium chloride and1-eicosylpyridinium bromide, a phosphonium salt represented by thegeneral formula (III),

wherein, R⁶ to R⁹, which may be the same or different, each represent asubstituted or unsubstituted C₁ to C₂₀ alkyl, alkenyl, aryl or benzyl;and Z⁻ represents a monovalent anion, tetraethylphosphonium chloride,tetraethylphosphonium bromide, tributylmethylphosphonium chloride,tributylmethylphosphonium bromide, tributylmethylphosphonium iodide,tetrabutylphosphonium chloride, tetrabutylphosphonium bromide,tetra-n-octylphosphonium chloride, tetra-n-octylphosphonium bromide,tributyldodecylphosphonium chloride, tributyldodecylphosphonium bromide,tributylhexadecylphosphonium chloride, tributylhexadecylphosphoniumbromide, methyltriphenylphosphonium chloride, methyltriphenylphosphoniumbromide, methyltriphenylphosphonium iodide, tetraphenylphosphoniumchloride and tetraphenylphosphonium bromide and sodium dodecyl sulfate.4. The method of claim 1, further comprising use of one or more buffersselected from the group consisting of: a borate buffer, a Tris-HClbuffer, a phosphate buffer, a citrate buffer, a fumarate buffer, aglutarate buffer, a citraconate buffer, a mesaconate buffer, a malonatebuffer, a tartrate buffer, a succinate buffer, an adipate buffer, ACES(N-(2-acetamido)-2-aminoethanesulfonic acid) buffer, BES(N,N-bis(2-hydroxyethyl)-2-amino-ethanesulfonic acid) buffer, Bicin(N,N-bis(2-hydroxyethyl)glycine) buffer, Bis-Tris(bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane) buffer, EPPS(4-(2-hydroxyethyl)-1-piperazine-propanesulfonic acid) buffer, HEPPSO(N-(hydroxyethyl)piperazine-N′-2-hydroxypropanesulfonic acid) buffer,MES (2-(n-morpholino)ethanesulfonic acid) buffer, MOPS(3-(N-morpholino)propanesulfonic acid) buffer, MOPSO(2-hydroxy-3-morpholino-propanesulfonate) buffer, PIPES(piperazine-N,N′-bis(2-ethanesulfonic acid)) buffer, POPSO(piperazine-1,4-bis(2-hydroxypropanesulfonic acid)) buffer, TAPS(N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid) buffer, TAPSO(3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid)buffer, TES (N-tris(hydroxymethyl)methyl-2-amino-ethanesulfonic acid)buffer, Tricine (N-Tris(hydroxymethyl)methylglycine) buffer and acombination thereof, a phosphate buffer having a final concentration inthe measurement solution of 100 mM or higher, a citrate buffer having afinal concentration in the measurement solution of 10 mM or higher, MES(2-(n-morpholino)ethanesulfonic acid) buffer having a finalconcentration in the measurement solution of 150 mM or higher, MOPS(3-(n-morpholino)propanesulfonic acid) buffer having a finalconcentration in the measurement solution of 100 mM or higher MOPSO(2-hydroxy-3-morpholino-propanesulfonic acid) buffer having a finalconcentration in the measurement solution of 100 mM or higher, and ACES(N-(2-acetamido)-2-aminoethanesulfonic acid) buffer having a finalconcentration in the measurement solution of 200 mM or higher.
 5. Themethod of claim 1, further comprising use of one or more stabilizersselected from the group consisting of phosphoric acid, a tricarboxylicacid, a dicarboxylic acid, a monocarboxylic acid, a compound representedby the formula (IV)

wherein, n may be 0, 1, 2 or 3; and R¹⁰ each independently represents H,OH, —CH₂OH or —COOH, ammonium sulfate, citric acid, fumaric acid,glutaric acid, citraconic acid, mesaconic acid, malonic acid, tartaricacid, succinic acid, adipic acid, maleic acid, malic acid, acetic acid,MES, MOPS, MOPSO, and a combination thereof.
 6. The method of claim 4,further comprising use of one or more stabilizers selected from thegroup consisting of phosphoric acid, a tricarboxylic acid, adicarboxylic acid, a monocarboxylic acid, a compound represented by thefollowing formula

wherein, n may be 0, 1, 2 or 3; and R¹⁰ each independently represents H,OH, —CH₂OH or —COOH, ammonium sulfate, citric acid, fumaric acid,glutaric acid, citraconic acid, mesaconic acid, malonic acid, tartaricacid, succinic acid, adipic acid, maleic acid, malic acid, acetic acid,MES, MOPS, MOPSO, and a combination thereof.
 7. A method for maintainingthe residual activity of an amadoriase contained in reagents formeasuring glycated hemoglobin in the presence of an ionic surfactant orfor alleviating reduction of the residual activity of an amadoriasecontained in reagents for measuring glycated hemoglobin in the presenceof an ionic surfactant, said method comprising using one or more ionicsurfactant, an amadoriase, and a stabilizer that is capable ofmaintaining the residual activity of an amadoriase in the presence of anionic surfactant or capable of alleviating reduction of the residualactivity of an amadoriase in the presence of an ionic surfactant,wherein said stabilizer is selected from the group consisting ofphosphoric acid, a tricarboxylic acid, a dicarboxylic acid, amonocarboxylic acid, a compound represented by the formula (IV)

wherein, n may be 0, 1, 2 or 3; and R¹⁰ each independently represents H,OH, —CH₂OH or —COOH, ammonium sulfate, citric acid, fumaric acid,glutaric acid, citraconic acid, mesaconic acid, malonic acid, tartaricacid, succinic acid, adipic acid, maleic acid, malic acid, acetic acid,MES, MOPS, MOPSO, and a combination thereof.
 8. The method of claim 7,wherein the stabilizer is one or more stabilizers selected from thegroup consisting of phosphoric acid having a final concentration in themeasurement solution of 2 mM or higher, citric acid having a finalconcentration in the measurement solution of 0.2 mM or higher, malicacid having a final concentration in the measurement solution of 2 mM orhigher, maleic acid having a final concentration in the measurementsolution of 2 mM or higher, citraconic acid having a final concentrationin the measurement solution of 2 mM or higher, malonic acid having afinal concentration in the measurement solution of 2 mM or higher,glutaric acid having a final concentration in the measurement solutionof 2 mM or higher, tartaric acid having a final concentration in themeasurement solution of 2 mM or higher, acetic acid having a finalconcentration in the measurement solution of 10 mM or higher, MES(2-(n-morpholino)ethanesulfonic acid) having a final concentration inthe measurement solution of 10 mM or higher, MOPS(3-(n-morpholino)propanesulfonic acid) having a final concentration inthe measurement solution of 10 mM or higher, MOPSO(2-hydroxy-3-morpholinopropanesulfonic acid) having a finalconcentration in the measurement solution of 10 mM or higher, ammoniumsulfate having a final concentration in the measurement solution of 2 mMor higher and a combination thereof
 9. A method for maintaining theresidual activity of an amadoriase contained in reagents for measuringglycated hemoglobin in the presence of an ionic surfactant or foralleviating reduction of the residual activity of an amadoriasecontained in reagents for measuring glycated hemoglobin in the presenceof an ionic surfactant, said method comprising using one or more ionicsurfactant, an amadoriase, and a buffer that is capable of maintainingthe residual activity of an amadoriase in the presence of an ionicsurfactant or is capable of alleviating reduction of the residualactivity of an amadoriase in the presence of an ionic surfactant,wherein said buffer is selected from the group consisting of a boratebuffer, a Tris-HCl buffer, a phosphate buffer, a citrate buffer, afumarate buffer, a glutarate buffer, a citraconate buffer, a mesaconatebuffer, a malonate buffer, a tartrate buffer, a succinate buffer, anadipate buffer, ACES (N-(2-acetamido)-2-aminoethanesulfonic acid)buffer, BES (N,N-bis(2-hydroxyethyl)-2-amino-ethanesulfonic acid)buffer, Bicin (N,N-bis(2-hydroxyethyl)glycine) buffer, Bis-Tris(bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane) buffer, EPPS(4-(2-hydroxyethyl)-1-piperazine-propanesulfonic acid) buffer, HEPPSO(N-(hydroxyethyl)piperazine-N′-2-hydroxypropanesulfonic acid) buffer,MES (2-(n-morpholino)ethanesulfonic acid) buffer, MOPS(3-(N-morpholino)propanesulfonic acid) buffer, MOPSO(2-hydroxy-3-morpholino-propanesulfonate) buffer, PIPES(piperazine-N,N′-bis(2-ethanesulfonic acid)) buffer, POPSO(piperazine-1,4-bis(2-hydroxypropanesulfonic acid)) buffer, TAPS(N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid) buffer, TAPSO(3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid)buffer, TES (N-tris(hydroxymethyl)methyl-2-amino-ethanesulfonic acid)buffer, Tricine (N-Tris(hydroxymethyl)methylglycine) buffer and acombination thereof.
 10. The method of claim 9, wherein the buffercomprises a buffer selected from the group consisting of a phosphatebuffer having a final concentration in the measurement solution of 100mM or higher, a citrate buffer having a final concentration in themeasurement solution of 10 mM or higher, MES(2-(n-morpholino)ethanesulfonic acid) buffer having a finalconcentration in the measurement solution of 150 mM or higher, MOPS(3-(n-morpholino)propanesulfonic acid) buffer having a finalconcentration in the measurement solution of 100 mM or higher MOPSO(2-hydroxy-3-morpholino-propanesulfonic acid) buffer having a finalconcentration in the measurement solution of 100 mM or higher, and ACES(N-(2-acetamido)-2-aminoethanesulfonic acid) buffer having a finalconcentration in the measurement solution of 200 mM or higher.
 11. Themethod of claim 7, further comprising using one or more buffer that iscapable of maintaining the residual activity of an amadoriase in thepresence of an ionic surfactant or is capable of alleviating reductionof the residual activity of an amadoriase in the presence of an ionicsurfactant, wherein said buffer is selected from the group consisting ofa borate buffer, a Tris-HCl buffer, a phosphate buffer, a citratebuffer, a fumarate buffer, a glutarate buffer, a citraconate buffer, amesaconate buffer, a malonate buffer, a tartrate buffer, a succinatebuffer, an adipate buffer, ACES (N-(2-acetamido)-2-aminoethanesulfonicacid) buffer, BES (N,N-bis(2-hydroxyethyl)-2-amino-ethanesulfonic acid)buffer, Bicin (N,N-bis(2-hydroxyethyl)glycine) buffer, Bis-Tris(bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane) buffer, EPPS(4-(2-hydroxyethyl)-1-piperazine-propanesulfonic acid) buffer, HEPPSO(N-(hydroxyethyl)piperazine-N′-2-hydroxypropanesulfonic acid) buffer,MES (2-(n-morpholino)ethanesulfonic acid) buffer, MOPS(3-(N-morpholino)propanesulfonic acid) buffer, MOPSO(2-hydroxy-3-morpholino-propanesulfonate) buffer, PIPES(piperazine-N,N′-bis(2-ethanesulfonic acid)) buffer, POPSO(piperazine-1,4-bis(2-hydroxypropanesulfonic acid)) buffer, TAPS(N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid) buffer, TAPSO(3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid)buffer, TES (N-tris(hydroxymethyl)methyl-2-amino-ethanesulfonic acid)buffer, Tricine (N-Tris(hydroxymethyl)methylglycine) buffer and acombination thereof.
 12. A composition for carrying out the method ofclaim 7, said composition comprising said one or more ionic surfactant,amadoriase, and stabilizer of claim
 7. 13. A composition for carryingout the method of claim 9, said composition comprising said one or moreionic surfactant, amadoriase, and buffer of claim 9.